Treatment of cancer using humanized anti-EGFRvIII chimeric antigen receptor

ABSTRACT

The invention provides compositions and methods for treating diseases associated with expression of EGFRvIII. The invention also relates to chimeric antigen receptor (CAR) specific to EGFRvIII, vectors encoding the same, and recombinant T cells comprising the anti-EGFRvIII CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises an anti-EGFRvIII binding domain.

This application claims priority to U.S. Ser. No. 61/888,255 filed Oct.8, 2013 and U.S. Ser. No. 61/767,071, filed Feb. 20, 2013, the entirecontents of each of these applications is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 2R01 NS055140 and1P01 CA1322714 awarded by the National Institute of Health (NIH). Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 11, 2014, isnamed N2067-7000WO_SL.txt and is 224,156 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the use of T cells engineeredto express a Chimeric Antigen Receptor (CAR) to treat a diseaseassociated with expression of Epidermal Growth Factor Receptor III.

BACKGROUND OF THE INVENTION

Although the central nervous system (CNS) is often considered to beimmunologically privileged (Okada et al., 2009, Crit Rev Immunol29:1-42), recent vaccine studies in patients with malignant gliomademonstrated positive results (Aguilar et al., 2012, Curr Treat OptionsOncol 13:437-450; Ruzevick, et al., 2012, Neurosurg Clin N Am23:459-470; 15; and Okada et al., 2011, J Clin Oncol 29:330-336).However, vaccine efficacy, which relies on intact host-immune activity,can suffer from systemic suppression of immunity due to tumor expressionof immunosuppressive cytokines as well as chemo- and radiotherapy. Onthe other hand, adoptive cell transfer (ACT) therapy with autologousT-cells, especially with T-cells transduced with Chimeric AntigenReceptors (CARs), has shown promise in pilot hematologic cancer trials(Kalos et al., 2011, Sci Transl Med 3(95):95ra73; and Porter et al.,2011, New England Journal of Medicine 365:725-733).

Enhanced expression of epidermal growth factor receptor (EGFR) isfrequently detected in a variety of carcinomas, including breast, lung,head and neck, as well as glioblastoma. Spontaneous rearrangementswithin the EGF receptor gene were first identified in primary humanglioblastoma tumors, and in nearly all cases the alterations have beenreported in tumors with EGFR amplification. Three different types ofmutants result from these rearrangements. The most common of these isthe Type III EGF deletion-mutant receptor (EGFRvIII), which ischaracterized by the deletion of exons 2-7 in the EGFR mRNA. Thesedeletions correspond to cDNA nucleotides 275-1075, which encode aminoacids 6-276, presumably through alternative splicing or rearrangements.Deletion of 801 bp within the extracellular domain of the EGFR genecauses an in-frame truncation of the normal EGFR protein, resulting in a145-kDa receptor, thereby creating a tumor specific and immunogenicepitope (reviewed in Hatanpaa et al., 2010, Neoplasia 12:675-684; Mukasaet al., 2010, Proc Natl Acad Sci USA 107:2616-2621). EGFRvIII expressionhas been seen in many tumor types, including glioblastoma multiforme(GBM), but is rarely observed in normal tissue. EGFRvIII is expressed in24% to 67% of GBM cases, and in patients surviving ≧1 year, theexpression of EGFRvIII is an independent negative prognostic indicator(Heimberger et al., 2005, Clin. Cancer Res. 11:1462-1466; Heimberger etal., 2005, J Transl. Med 3:38).

SUMMARY OF THE INVENTION

The invention provides, among other things, compositions and methods forcontrolling an immune response in patients by providing optimized and/orhumanized antibodies or antibody fragments (e.g., scFv) that bindEpidermal Growth Factor Receptor III (EGFRvIII) integrated into aChimeric Antigen Receptor (CAR) construct. In some embodiments, theinvention pertains to the use of T cells engineered to express anantibody or antibody fragment that bind EGFRvIII, e.g., a humanizedantibody or antibody fragment that binds EGFRvIII, integrated into a CARto treat a cancer associated with expression of EGFRvIII. In someaspects, the invention pertains to adoptive cell transfer that may beparticularly suitable for patients with glioma because the specificity,number, and functional phenotype of cells prepared ex vivo can bemanipulated and controlled far better than native T-cells induced by invivo immunization.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises an antibody or antibody fragment whichincludes an anti-EGFRvIII binding domain (e.g., a humanized antibody orantibody fragment that specifically binds to EGFRvIII), a transmembranedomain, and an intracellular signaling domain (e.g., an intracellularsignaling domain comprising a costimulatory domain and/or a primarysignaling domain). In one embodiment, the CAR comprises an antibody orantibody fragment which includes an anti-EGFRvIII binding domaindescribed herein (e.g., a humanized antibody or antibody fragment thatspecifically binds to EGFRvIII as described herein), a transmembranedomain described herein, and an intracellular signaling domain describedherein (e.g., an intracellular signaling domain comprising acostimulatory domain and/or a primary signaling domain).

In one embodiment, the encoded anti-EGFRvIII binding domain comprisesone or more (e.g., all three) light chain complementary determiningregion 1 (LC CDR1), light chain complementary determining region 2 (LCCDR2), and light chain complementary determining region 3 (LC CDR3) ofan anti-EGFRvIII binding domain described herein, and one or more (e.g.,all three) heavy chain complementary determining region 1 (HC CDR1),heavy chain complementary determining region 2 (HC CDR2), and heavychain complementary determining region 3 (HC CDR3) of an anti-EGFRvIIIbinding domain described herein, e.g., a humanized anti-EGFRvIII bindingdomain comprising one or more, e.g., all three, LC CDRs and one or more,e.g., all three, HC CDRs. In one embodiment, the encoded anti-EGFRvIIIbinding domain comprises a light chain variable region described herein(e.g., in Table 2 or SEQ ID NO:11) and/or a heavy chain variable regiondescribed herein (e.g., in Table 2 or SEQ ID NO:11). In one embodiment,the encoded anti-EGFRvIII binding domain is a scFv comprising a lightchain and a heavy chain of an amino acid sequence of Table 2 or SEQ IDNO:11. In an embodiment, the anti-EGFRvIII binding domain (e.g., anscFv) comprises: a light chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions) of an amino acid sequence of a light chain variableregion provided in Table 2 or SEQ ID NO:11, or a sequence with 95-99%identity with an amino acid sequence of Table 2 or SEQ ID NO:11; and/ora heavy chain variable region comprising an amino acid sequence havingat least one, two or three modifications (e.g., substitutions) but notmore than 30, 20 or 10 modifications (e.g., substitutions) of an aminoacid sequence of a heavy chain variable region provided in Table 2 orSEQ ID NO:11, or a sequence with 95-99% identity to an amino acidsequence of Table 2 or SEQ ID NO:11. In one embodiment, theanti-EGFRvIII binding domain comprises a sequence selected from a groupconsisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56,SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ IDNO:86, or a sequence with 95-99% identify thereof. In one embodiment,the nucleic acid sequence encoding the anti-EGFRvIII binding domaincomprises a sequence of SEQ ID NO:68. In one embodiment, the nucleicacid sequence encoding the anti-EGFRvIII binding domain comprises asequence selected from a group consisting of SEQ ID NO:39, SEQ ID NO:45,SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:69, SEQ ID NO:75,SEQ ID NO:81, and SEQ ID NO:98, or a sequence with 95-99% identifythereof. In one embodiment, the encoded anti-EGFRvIII binding domain isa scFv, and a light chain variable region comprising an amino acidsequence described herein, e.g., in Table 2 or SEQ ID NO:11, is attachedto a heavy chain variable region comprising an amino acid sequencedescribed herein, e.g., in Table 2 or SEQ ID NO:11, via a linker, e.g.,a linker described herein. In one embodiment, the encoded anti-EGFRvIIIbinding domain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4,5, or 6, preferably 4 (SEQ ID NO: 110). The light chain variable regionand heavy chain variable region of a scFv can be, e.g., in any of thefollowing orientations: light chain variable region-linker-heavy chainvariable region or heavy chain variable region-linker-light chainvariable region.

In one embodiment, the encoded CAR includes a transmembrane domain thatcomprises a transmembrane domain of a protein selected from the groupconsisting of the alpha, beta or zeta chain of the T-cell receptor,CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the encodedtransmembrane domain comprises a sequence of SEQ ID NO: 15. In oneembodiment, the encoded transmembrane domain comprises an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:15, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO:15. In oneembodiment, the nucleic acid sequence encoding the transmembrane domaincomprises a sequence of SEQ ID NO:8, or a sequence with 95-99% identifythereof.

In one embodiment, the encoded anti-EGFRvIII binding domain is connectedto the transmembrane domain by a hinge region, e.g., a hinge regiondescribed herein. In one embodiment, the encoded hinge region comprisesSEQ ID NO:14 or SEQ ID NO:104 or SEQ ID NO:106 or SEQ ID NO:108, or asequence with 95-99% identity thereof. In one embodiment, the nucleicacid sequence encoding the hinge region comprises a sequence of SEQ IDNO:7, or SEQ ID NO:105 or SEQ ID NO:107 or SEQ ID NO:109 or a sequencewith 95-99% identify thereof.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein. In one embodiment, the encoded costimulatory domaincomprises a functional signaling domain of a protein selected from thegroup consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, theencoded costimulatory domain comprises a sequence of SEQ ID NO:16. Inone embodiment, the encoded costimulatory domain comprises a sequence ofSEQ ID NO:102. In one embodiment, the encoded costimulatory domaincomprises an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO:16 or SEQ ID NO:102, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO:16 or SEQ ID NO:102. In one embodiment, thenucleic acid sequence encoding the costimulatory domain comprises asequence of SEQ ID NO:9, or a sequence with 95-99% identify thereof.

In one embodiment, the isolated nucleic acid molecule further comprisesa sequence encoding an intracellular signaling domain, e.g., anintracellular signaling domain described herein. In one embodiment, theencoded intracellular signaling domain comprises a functional signalingdomain of 4-1BB and/or a functional signaling domain of CD3 zeta. In oneembodiment, the encoded intracellular signaling domain comprises afunctional signaling domain of CD27 and/or a functional signaling domainof CD3 zeta. In one embodiment, the encoded intracellular signalingdomain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 102 and/orthe sequence of SEQ ID NO: 17 or SEQ ID NO:99. In one embodiment, theintracellular signaling domain comprises an amino acid sequence havingat least one, two or three modifications (e.g., substitutions) but notmore than 20, 10 or 5 modifications (e.g., substitutions) of an aminoacid sequence of SEQ ID NO:16 and/or an amino acid sequence of SEQ IDNO:17 or SEQ ID NO:99, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO:16 or SEQ ID NO:102 and/or an amino acidsequence of SEQ ID NO:17 or SEQ ID NO:99. In one embodiment, the encodedintracellular signaling domain comprises the sequence of SEQ ID NO: 16or SEQ ID NO:102 and the sequence of SEQ ID NO: 17 or SEQ ID NO:99,wherein the sequences comprising the intracellular signaling domain areexpressed in the same frame and as a single polypeptide chain. In oneembodiment, the nucleic acid sequence encoding the intracellularsignaling domain comprises a sequence of SEQ ID NO:9 or SEQ ID NO:103,or a sequence with 95-99% identify thereof, and/or a sequence of SEQ IDNO:10 or SEQ ID NO:100, or a sequence with 95-99% identity thereof.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence, e.g., aleader sequence described herein, e.g., of SEQ ID NO: 13, ananti-EGFRvIII binding domain described herein, e.g., an anti-EGFRvIIIbinding domain comprising a LC CDR1, a LC CDR2, a LC CDR3, a HC CDR1, aHC CDR2 and a HC CDR3 described herein, e.g., an anti-EGFRvIII bindingdomain described in Table 2 or SEQ ID NO:11, or a sequence with 95-99%identify thereof, a hinge region described herein, e.g., of SEQ ID NO:14or SEQ ID NO:104 or SEQ ID NO:106 or SEQ ID NO:108, a transmembranedomain described herein, e.g., having a sequence of SEQ ID NO: 15, andan intracellular signaling domain, e.g., an intracellular signalingdomain described herein. In one embodiment, the encoded intracellularsignaling domain comprises a costimulatory domain, e.g., a costimulatorydomain described herein, e.g., a 4-1BB costimulatory domain having asequence of SEQ ID NO:16, and/or a primary signaling domain, e.g., aprimary signaling domain described herein, e.g., a CD3 zeta stimulatorydomain having a sequence of SEQ ID NO:17 or SEQ ID NO:99. In oneembodiment, the encoded intracellular signaling domain comprises acostimulatory domain, e.g., a costimulatory domain described herein,e.g., a CD27 costimulatory domain having a sequence of SEQ ID NO:102,and/or a primary signaling domain, e.g., a primary signaling domaindescribed herein, e.g., a CD3 zeta stimulatory domain having a sequenceof SEQ ID NO:17 or SEQ ID NO:99. In one embodiment, the encodedintracellular signaling domain comprises a costimulatory domain, e.g., acostimulatory domain described herein, e.g., a 4-1BB costimulatorydomain having a sequence of SEQ ID NO:16, and a primary signalingdomain, e.g., a primary signaling domain described herein, e.g., a CD3zeta stimulatory domain having a sequence of SEQ ID NO:17 or SEQ IDNO:99. In one embodiment, the encoded intracellular signaling domaincomprises a costimulatory domain, e.g., a costimulatory domain describedherein, e.g., a CD27 costimulatory domain having a sequence of SEQ IDNO:102, and a primary signaling domain, e.g., a primary signaling domaindescribed herein, e.g., a CD3 zeta stimulatory domain having a sequenceof SEQ ID NO:17 or SEQ ID NO:99. In one embodiment, the isolated nucleicacid molecule encoding the CAR construct includes a leader sequenceencoded by the nucleic acid sequence of SEQ ID NO:6, or a sequence with95-99% identity thereto. In one embodiment, the isolated nucleic acidmolecule encoding the CAR construct includes an anti-EGFR binding domainsequence encoded by the nucleic acid sequence of SEQ ID NO:39, SEQ IDNO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:69, SEQ IDNO:75, SEQ ID NO:81, or SEQ ID NO:98, or a sequence with 95-99% identitythereto. In one embodiment, the isolated nucleic acid molecule encodingthe CAR construct includes an anti-EGFR binding domain sequence encodedby the nucleic acid sequence of SEQ ID NO:69, or a sequence with 95-99%identity thereto. In one embodiment, the isolated nucleic acid moleculeencoding the CAR construct includes an anti-EGFR binding domain sequenceencoded by the nucleic acid sequence of SEQ ID NO:4, or a sequence with95-99% identity thereto. In one embodiment, the isolated nucleic acidmolecule encoding the CAR construct includes a transmembrane sequenceencoded by the nucleic acid sequence of SEQ ID NO:8, or a sequence with95-99% identity thereto. In one embodiment, the isolated nucleic acidmolecule encoding the CAR construct includes an intracellular signalingdomain sequence encoded by the nucleic acid sequence of SEQ ID NO:9, ora sequence with 95-99% identity thereto and/or a nucleic acid sequenceof SEQ ID NO:10, or a sequence with 95-99% identity thereto.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid encoding a CAR amino acid sequence of SEQ IDNO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ IDNO:73, SEQ ID NO:79, SEQ ID NO:85, or SEQ ID NO:90, or an amino acidsequence having at least one, two, three, four, five, 10, 15, 20 or 30modifications (e.g., substitutions) but not more than 60, 50 or 40modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ IDNO:73, SEQ ID NO:79, SEQ ID NO:85, or SEQ ID NO:90, or an amino acidsequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an aminoacid sequence of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61,SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, or SEQ ID NO:90.In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid encoding a CAR amino acid sequence of SEQ IDNO:1, or SEQ ID NO:2, or an amino acid sequence having at least one,two, three, four, five, 10, 15, 20 or 30 modifications (e.g.,substitutions) but not more than 60, 50 or 40 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:1, or SEQ ID NO:2,or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

In one embodiment, the isolated nucleic acid molecule comprises (e.g.,consists of) a nucleic acid sequence of SEQ ID NO:42, SEQ ID NO:48, SEQID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:78, SEQ IDNO:84, or SEQ ID NO:89 or a nucleic acid sequence having 85%, 90%, 95%,96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ IDNO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ IDNO:72, SEQ ID NO:78, SEQ ID NO:84, or SEQ ID NO:89. In one embodiment,the isolated nucleic acid molecule comprises (e.g., consists of) anucleic acid sequence of SEQ ID NO:18, or SEQ ID NO:19, or a nucleicacid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to anucleic acid sequence of SEQ ID NO:18 or SEQ ID NO:19.

In one aspect, the invention pertains to an isolated nucleic acidmolecule encoding an anti-EGFRvIII binding domain, wherein theanti-EGFRvIII binding domain comprises one or more (e.g., all three)light chain complementary determining region 1 (LC CDR1), light chaincomplementary determining region 2 (LC CDR2), and light chaincomplementary determining region 3 (LC CDR3) of an anti-EGFRvIII bindingdomain described herein, and one or more (e.g., all three) heavy chaincomplementary determining region 1 (HC CDR1), heavy chain complementarydetermining region 2 (HC CDR2), and heavy chain complementarydetermining region 3 (HC CDR3) of an anti-EGFRvIII binding domaindescribed herein, e.g., a humanized anti-EGFRvIII binding domaincomprising one or more, e.g., all three, LC CDRs and one or more, e.g.,all three, HC CDRs. In one embodiment, the encoded anti-EGFRvIII bindingdomain comprises a light chain variable region described herein (e.g.,in SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80) and/or a heavy chainvariable region described herein (e.g., in SEQ ID NO:38, 44, 50, 56, 62,68, 74 or 80). In one embodiment, the encoded anti-EGFRvIII bindingdomain is a scFv comprising a light chain and a heavy chain of an aminoacid sequence of in SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80. In anembodiment, the anti-EGFRvIII binding domain (e.g., an scFv) comprises:a light chain variable region comprising an amino acid sequence havingat least one, two or three modifications (e.g., substitutions) but notmore than 30, 20 or 10 modifications (e.g., substitutions) of an aminoacid sequence of a light chain variable region provided in SEQ ID NO:38,44, 50, 56, 62, 68, 74 or 80, or a sequence with 95-99% identity with anamino acid sequence of SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80;and/or a heavy chain variable region comprising an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions) of anamino acid sequence of a heavy chain variable region provided in SEQ IDNO:38, 44, 50, 56, 62, 68, 74 or 80, or a sequence with 95-99% identityto an amino acid sequence in SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80.In one embodiment, the anti-EGFRvIII binding domain comprises a sequenceselected from a group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ IDNO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ IDNO:80, and SEQ ID NO:86, or a sequence with 95-99% identify thereof. Inone embodiment, the nucleic acid sequence encoding the anti-EGFRvIIIbinding domain comprises a sequence selected from a group consisting ofSEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63,SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, and SEQ ID NO:98, or asequence with 95-99% identify thereof. In one embodiment, the encodedanti-EGFRvIII binding domain is a scFv, and a light chain variableregion comprising an amino acid sequence described herein, e.g., inTable 2, is attached to a heavy chain variable region comprising anamino acid sequence described herein, e.g., in Table 2, via a linker,e.g., a linker described herein. In one embodiment, the encodedanti-EGFRvIII binding domain includes a (Gly₄-Ser)n linker, wherein n is1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 110). The light chainvariable region and heavy chain variable region of a scFv can be, e.g.,in any of the following orientations: light chain variableregion-linker-heavy chain variable region or heavy chain variableregion-linker-light chain variable region.

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ IDNO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85 and SEQ IDNO:90, or a sequence with 95-99% identify thereof. In one embodiment,the isolated polypeptide comprises a sequence of SEQ ID NO:73, or asequence with 95-99% identify thereof. In one embodiment, the isolatedpolypeptide comprises a sequence of SEQ ID NO:79, or a sequence with95-99% identify thereof.

In another aspect, the invention pertains to an isolated chimericantigen receptor (CAR) molecule comprising an anti-EGFRvIII bindingdomain (e.g., a humanized antibody or antibody fragment thatspecifically binds to EGFRvIII), a transmembrane domain, and anintracellular signaling domain (e.g., an intracellular signaling domaincomprising a costimulatory domain and/or a primary signaling domain). Inone embodiment, the CAR comprises an antibody or antibody fragment whichincludes an anti-EGFRvIII binding domain described herein (e.g., ahumanized antibody or antibody fragment that specifically binds toEGFRvIII as described herein), a transmembrane domain described herein,and an intracellular signaling domain described herein (e.g., anintracellular signaling domain comprising a costimulatory domain and/ora primary signaling domain described herein).

In one embodiment, the anti-EGFRvIII binding domain comprises one ormore (e.g., all three) light chain complementary determining region 1(LC CDR1), light chain complementary determining region 2 (LC CDR2), andlight chain complementary determining region 3 (LC CDR3) of ananti-EGFRvIII binding domain described herein, and one or more (e.g.,all three) heavy chain complementary determining region 1 (HC CDR1),heavy chain complementary determining region 2 (HC CDR2), and heavychain complementary determining region 3 (HC CDR3) of an anti-EGFRvIIIbinding domain described herein, e.g., a humanized anti-EGFRvIII bindingdomain comprising one or more, e.g., all three, LC CDRs and one or more,e.g., all three, HC CDRs. In one embodiment, the anti-EGFRvIII bindingdomain comprises a light chain variable region described herein (e.g.,in Table 2 or SEQ ID NO:11) and/or a heavy chain variable regiondescribed herein (e.g., in Table 2 or SEQ ID NO:11). In one embodiment,the anti-EGFRvIII binding domain is a scFv comprising a light chain anda heavy chain of an amino acid sequence listed in Table 2 or SEQ IDNO:11. In an embodiment, the anti-EGFRvIII binding domain (e.g., anscFv) comprises: a light chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions) of an amino acid sequence of a light chain variableregion provided in Table 2 or SEQ ID NO:11, or a sequence with 95-99%identity with an amino acid sequence provided in Table 2 or SEQ IDNO:11; and/or a heavy chain variable region comprising an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 30, 20 or 10 modifications (e.g.,substitutions) of an amino acid sequence of a heavy chain variableregion provided in Table 2 or SEQ ID NO:11, or a sequence with 95-99%identity to an amino acid sequence provided in Table 2 or SEQ ID NO:11.In one embodiment, the anti-EGFRvIII binding domain comprises a sequenceselected from a group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ IDNO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ IDNO:80, and SEQ ID NO:86, or a sequence with 95-99% identify thereof. Inone embodiment, the anti-EGFRvIII binding domain is a scFv, and a lightchain variable region comprising an amino acid sequence describedherein, e.g., in Table 2 or SEQ ID NO:11, is attached to a heavy chainvariable region comprising an amino acid sequence described herein,e.g., in Table 2 or SEQ ID NO:11, via a linker, e.g., a linker describedherein. In one embodiment, the anti-EGFRvIII binding domain includes a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 110). The light chain variable region and heavy chain variableregion of a scFv can be, e.g., in any of the following orientations:light chain variable region-linker-heavy chain variable region or heavychain variable region-linker-light chain variable region.

In one embodiment, the isolated CAR molecule comprises a transmembranedomain of a protein selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137and CD154. In one embodiment, the transmembrane domain comprises asequence of SEQ ID NO:15. In one embodiment, the transmembrane domaincomprises an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 20, 10 or 5modifications (e.g., substitutions) of an amino acid sequence of SEQ IDNO:15, or a sequence with 95-99% identity to an amino acid sequence ofSEQ ID NO:15.

In one embodiment, the anti-EGFRvIII binding domain is connected to thetransmembrane domain by a hinge region, e.g., a hinge region describedherein. In one embodiment, the encoded hinge region comprises SEQ IDNO:14 or SEQ ID NO:104 or SEQ ID NO:106 or SEQ ID NO:108, or a sequencewith 95-99% identity thereof.

In one embodiment, the isolated CAR molecule further comprises asequence encoding a costimulatory domain, e.g., a costimulatory domaindescribed herein. In one embodiment, the costimulatory domain comprisesa functional signaling domain of a protein selected from the groupconsisting of OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18) and4-1BB (CD137). In one embodiment, the costimulatory domain comprises asequence of SEQ ID NO:16 or SEQ ID NO:102. In one embodiment, thecostimulatory domain comprises an amino acid sequence having at leastone, two or three modifications (e.g., substitutions) but not more than20, 10 or 5 modifications (e.g., substitutions) of an amino acidsequence of SEQ ID NO:16 or SEQ ID NO:102, or a sequence with 95-99%identity to an amino acid sequence of SEQ ID NO:16 or SEQ ID NO:102. Inone embodiment, the isolated CAR molecule further comprises a sequenceencoding an intracellular signaling domain, e.g., an intracellularsignaling domain described herein. In one embodiment, the intracellularsignaling domain comprises a functional signaling domain of 4-1BB orCD27 and/or a functional signaling domain of CD3 zeta. In oneembodiment, the intracellular signaling domain comprises the sequence ofSEQ ID NO: 16 or SEQ ID NO:102 and/or the sequence of SEQ ID NO:17. Inone embodiment, the intracellular signaling domain comprises thesequence of SEQ ID NO: 16 or SEQ ID NO:102 and/or the sequence of SEQ IDNO:99. In one embodiment, the intracellular signaling domain comprisesan amino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:16 or SEQ IDNO:102 and/or an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:99, ora sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:16 or SEQ ID NO:102 and/or an amino acid sequence of SEQ ID NO:17 orSEQ ID NO:99. In one embodiment, the intracellular signaling domaincomprises the sequence of SEQ ID NO: 16 or SEQ ID NO:102 and thesequence of SEQ ID NO: 17 or SEQ ID NO:99, wherein the sequencescomprising the intracellular signaling domain are expressed in the sameframe and as a single polypeptide chain.

In one embodiment, the isolated CAR molecule further comprises a leadersequence, e.g., a leader sequence described herein. In one embodiment,the leader sequence comprises an amino acid sequence of SEQ ID NO: 13,or a sequence with 95-99% identity to an amino acid sequence of SEQ IDNO:13.

In another aspect, the invention pertains to an isolated CAR moleculecomprising a leader sequence, e.g., a leader sequence described herein,e.g., a leader sequence of SEQ ID NO: 13, or having 95-99% identitythereof, an anti-EGFRvIII binding domain described herein, e.g., ananti-EGFRvIII binding domain comprising a LC CDR1, a LC CDR2, a LC CDR3,a HC CDR1, a HC CDR2 and a HC CDR3 described herein, e.g., ananti-EGFRvIII binding domain described in Table 2 or SEQ ID NO:11, or asequence with 95-99% identify thereof, a hinge region, e.g., a hingeregion described herein, e.g., a hinge region of SEQ ID NO:14 or SEQ IDNO:104 or SEQ ID NO:106 or SEQ ID NO:108, or having 95-99% identitythereof, a transmembrane domain, e.g., a transmembrane domain describedherein, e.g., a transmembrane domain having a sequence of SEQ ID NO: 15or a sequence having 95-99% identity thereof, an intracellular signalingdomain, e.g., an intracellular signaling domain described herein (e.g.,an intracellular signaling domain comprising a costimulatory domainand/or a primary signaling domain). In one embodiment, the intracellularsignaling domain comprises a costimulatory domain, e.g., a costimulatorydomain described herein, e.g., a 4-1BB costimulatory domain having asequence of SEQ ID NO:16 or a CD27 costimulatory domain having asequence of SEQ ID NO:102, or having 95-99% identity thereof, and/or aprimary signaling domain, e.g., a primary signaling domain describedherein, e.g., a CD3 zeta stimulatory domain having a sequence of SEQ IDNO:17 or SEQ ID NO:99, or having 95-99% identity thereof. In oneembodiment, the intracellular signaling domain comprises a costimulatorydomain, e.g., a costimulatory domain described herein, e.g., a 4-1BBcostimulatory domain having a sequence of SEQ ID NO:16 or a CD27costimulatory domain having a sequence of SEQ ID NO:102, and/a primarysignaling domain, e.g., a primary signaling domain described herein,e.g., a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 orSEQ ID NO:99.

In one embodiment, the isolated CAR molecule comprises (e.g., consistsof) an amino acid sequence of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55,SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, orSEQ ID NO:90, or an amino acid sequence having at least one, two, three,four, five, 10, 15, 20 or 30 modifications (e.g., substitutions) but notmore than 60, 50 or 40 modifications (e.g., substitutions) of an aminoacid sequence of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61,SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, or SEQ ID NO:90,or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO:43, SEQ ID NO:49, SEQ IDNO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ IDNO:85, or SEQ ID NO:90. In one embodiment, the isolated CAR moleculecomprises (e.g., consists of) an amino acid sequence of SEQ ID NO:1,orSEQ ID NO:2, or an amino acid sequence having at least one, two,three, four, five, 10, 15, 20 or 30 modifications (e.g., substitutions)but not more than 60, 50 or 40 modifications (e.g., substitutions) of anamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or an amino acidsequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2. In one embodiment, theisolated CAR molecule comprises (e.g., consists of) an amino acidsequence of SEQ ID NO:73, or an amino acid sequence having at least one,two, three, four, five, 10, 15, 20 or 30 modifications (e.g.,substitutions) but not more than 60, 50 or 40 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO:73, or an aminoacid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to anamino acid sequence of SEQ ID NO:73. In one embodiment, the isolated CARmolecule comprises (e.g., consists of) an amino acid sequence of SEQ IDNO:79, or an amino acid sequence having at least one, two, three, four,five, 10, 15, 20 or 30 modifications (e.g., substitutions) but not morethan 60, 50 or 40 modifications (e.g., substitutions) of an amino acidsequence of SEQ ID NO:79, or an amino acid sequence having 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO:79.

In one aspect, the invention pertains to an anti-EGFRvIII binding domaincomprising one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of an anti-EGFRvIII binding domain described herein, and oneor more (e.g., all three) heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of ananti-EGFRvIII binding domain described herein, e.g., a humanizedanti-EGFRvIII binding domain comprising one or more, e.g., all three, LCCDRs and one or more, e.g., all three, HC CDRs. In one embodiment, theanti-EGFRvIII binding domain comprises a light chain variable regiondescribed herein (e.g., in SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80)and/or a heavy chain variable region described herein (e.g. in SEQ IDNO:38, 44, 50, 56, 62, 68, 74 or 80). In one embodiment, theanti-EGFRvIII binding domain is a scFv comprising a light chain and aheavy chain of an amino acid sequence of in SEQ ID NO:38, 44, 50, 56,62, 68, 74 or 80. In an embodiment, the anti-EGFRvIII binding domain(e.g., an scFv) comprises: a light chain variable region comprising anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions) of an amino acid sequence of a light chainvariable region provided, in SEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80or a sequence with 95-99% identity with an amino acid sequence in SEQ IDNO:38, 44, 50, 56, 62, 68, 74 or 80; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a heavychain variable region provided in SEQ ID NO:38, 44, 50, 56, 62, 68, 74or 80, or a sequence with 95-99% identity to an amino acid sequence inSEQ ID NO:38, 44, 50, 56, 62, 68, 74 or 80. In one embodiment, theanti-EGFRvIII binding domain comprises a sequence selected from a groupconsisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56,SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ IDNO:86, or a sequence with 95-99% identify thereof. In one embodiment,the anti-EGFRvIII binding domain is a scFv, and a light chain variableregion comprising an amino acid sequence described herein, e.g., inTable 2, is attached to a heavy chain variable region comprising anamino acid sequence described herein, e.g., in Table 2, via a linker,e.g., a linker described herein. In one embodiment, the anti-EGFRvIIIbinding domain includes a (Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4,5, or 6, preferably 4 (SEQ ID NO: 110). The light chain variable regionand heavy chain variable region of a scFv can be, e.g., in any of thefollowing orientations: light chain variable region-linker-heavy chainvariable region or heavy chain variable region-linker-light chainvariable region.

In another aspect, the invention pertains to a vector comprising anucleic acid molecule described herein, e.g., a nucleic acid moleculeencoding a CAR described herein. In one embodiment, the vector isselected from the group consisting of a DNA, a RNA, a plasmid, alentivirus vector, adenoviral vector, or a retrovirus vector.

In one embodiment, the vector is a lentivirus vector. In one embodiment,the vector further comprises a promoter. In one embodiment, the promoteris an EF-1 promoter. In one embodiment, the EF-1 promoter comprises asequence of SEQ ID NO: 97.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, e.g.,comprising about 150 adenosine bases (SEQ ID NO: 111). In oneembodiment, the nucleic acid sequence in the vector further comprises a3′UTR, e.g., a 3′ UTR described herein, e.g., comprising at least onerepeat of a 3′UTR derived from human beta-globulin.

In another aspect, the invention pertains to a cell comprising a vectordescribed herein. In one embodiment, the cell is a cell describedherein, e.g., a human T cell, e.g., a human T cell described herein. Inone embodiment, the human T cell is a CD8+ T cell.

In another aspect, the invention pertains to a method of making a cellcomprising transducing a cell described herein, e.g., a T cell describedherein, with a vector of comprising a nucleic acid encoding a CAR, e.g.,a CAR described herein.

The present invention also provides a method of generating a populationof RNA-engineered cells, e.g., cells described herein, e.g., T cells,transiently expressing exogenous RNA. The method comprises introducingan in vitro transcribed RNA or synthetic RNA into a cell, where the RNAcomprises a nucleic acid encoding a CAR molecule described herein.

In another aspect, the invention pertains to a method of providing ananti-tumor immunity in a mammal comprising administering to the mammalan effective amount of a cell expressing a CAR molecule, e.g., a cellexpressing a CAR molecule described herein. In one embodiment, the cellis an autologous T cell. In one embodiment, the cell is an allogeneic Tcell. In one embodiment, the mammal is a human.

In another aspect, the invention pertains to a method of treating amammal having a disease associated with expression of EGFRvIII (e.g., aproliferative disease, a precancerous condition, and a noncancer relatedindication associated with the expression of EGFRvIII) comprisingadministering to the mammal an effective amount of the cells expressinga CAR molecule, e.g., a CAR molecule described herein.

In one embodiment, the disease is a disease described herein. In oneembodiment, the disease associated with EGFRvIII is a glioblastoma. Inone embodiment, the disease associated with EGFRvIII is a cancer, e.g.,a cancer selected from the group consisting of glioblastoma multiforme(GBM), anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma,anaplastic oligodendroglioma, anaplastic ependymoma, choroid plexuscarcinoma, anaplastic ganglioglioma, pineoblastoma, medulloepithelioma,ependymoblastoma, medulloblastoma, supratentorial primitiveneuroectodermal tumor, atypical teratoid/rhabdoid tumor, lung cancer(e.g., non-small cell lung carcinomas) breast, prostate, ovarian,colorectal and bladder carcinoma and any combination thereof, andmetastases of any of the cancers.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat increases the efficacy of a cell expressing a CAR molecule, e.g.,an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat ameliorates one or more side effect associated with administrationof a cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with an agentthat treats the disease associated with EGFRvIII, e.g., an agentdescribed herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered at a dose and/or dosingschedule described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a first line treatmentfor the disease, e.g., the cancer, e.g., the cancer described herein. Inanother embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a second, third, fourthline treatment for the disease, e.g., the cancer, e.g., the cancerdescribed herein.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use as amedicament, e.g., as described herein.

In another aspect, the invention pertains to a the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use in thetreatment of a disease expressing EGFRvIII, e.g., a disease expressingEGFRvIII as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a series of schematic diagrams of lentiviral vectorsfor 3C10-CAR and miR-17-92. FIG. 1A depicts the 3C10-CAR expressingvector pELNS-3C10-CAR; and FIG. 1B depicts the miR-17-92-expressinglentiviral vector;

FIGS. 2A through 2C are a series of images showing the functionalexpression of lentivirally transduced 3C10-CAR and miR-17-92 in human Tcells. CD3+ T cells that were transduced with pELNS-3C10-CAR alone orboth pELNS-3C10-CAR and FG12-EF1a-miR-17/92;

FIGS. 3A through 3D are a series of images demonstrating thatco-expression of miR17-92 in CAR-T-cells confers resistance tosuppressive effects of TGF-β and TMZ. CAR-T-cells (open bars) and thoseco-transduced with miR-17-92 (closed bars) were co-cultured with an APCsexpressing EGFRvIII in the presence of indicated concentrations of TGF-βand TMZ;

FIGS. 4A and 4B are images depicting robust therapeutic effects ofCAR-T-cells in mice bearing U87-EGFRvIII tumors;

FIGS. 5A through 5C are a series of images demonstrating thatco-transduced miR-17-92 in CAR-T cells confers improved protectionagainst re-challenged glioma cells;

FIG. 6 is an image showing the comparison of the representative EGFRvIIICARs (SEQ ID NOS 1, 121, and 2, respectively, in order of appearance);

FIGS. 7A and 7B are images showing that human T-cells transduced withEGFRvIII CARs exhibited specific and potent lysis of EGFRvIII-expressingU87 human GBM cells (U87-EGFRvIII);

FIG. 8 is a graph showing that all anti-EGFRvIII CARTs clear tumorcells, but construct 3C10.BBz CART clears tumors most rapidly by day 7;

FIG. 9 is a table showing the VH and VL sequences of humanized EGFRvIII(SEQ ID NOS 122-127, respectively, in order of appearance);

FIG. 10 is a graph showing in vitro binding of soluble humanized scFvconstructs binding to EGFRvIII+cell line;

FIG. 11 is a graph showing in vitro binding of soluble humanized scFvconstructs binding to EGFR wild type cell line, with clone 73 (alsoreferred to as CAR6) and clone 74 (also referred to as CAR7) showing asafer profile;

FIGS. 12A and 12B are graphs comparing the specificity of murine CAR9and human CAR10 for EGFRvIII and wild type EGFR in transienttransfection of Jurkat cells and detection with Fc fusion proteins;

FIG. 13 is a graph showing primary T cell transduction of donor T cellswith the humanized EGFRvIII CAR constructs mCAR19 (control), CAR10,CAR9, and CAR6, stained with saturating amounts of EGFRvIII;

FIG. 14 is a graph showing the luciferase activity of humanized EGFRvIIICAR constructs by BHK-EGFRvIII but not wild type cells;

FIG. 15 is a graph showing that the humanized EGFRvIII CAR constructsproliferate in response to U87vIII challenge with no backgroundproliferation to wild type EGFR;

FIG. 16 is a graph showing that the humanized EGFRvIII CAR constructsproliferate in vitro in the presence of U87vIII challenge;

FIG. 17 is a graph showing a 4 hour 51-Chromium release tumor killingassay in which the humanized EGFRvIII CAR construct, 2173 (CAR6) andCAR9 specifically kills EGFRvIII expressing but not wild type EGFRcells; and

FIG. 18 is a graph showing progression of tumor size (cm³, upper leftpanel) and progression of tumor radiance average (p/s/cm²/sr, upperright panel), and Kaplan-meier survival curve (lower) in vivo in micetreated with CAR+ T cells transduced with the humanized EGFRvIII CARcontruct (CAR6).

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa recombinant polypeptide construct comprising at least an extracellularantigen binding domain, a transmembrane domain and a cytoplasmicsignaling domain (also referred to herein as “an intracellular signalingdomain”) comprising a functional signaling domain derived from astimulatory molecule as defined below. In one aspect, the stimulatorymolecule is the zeta chain associated with the T cell receptor complex.In one aspect, the cytoplasmic signaling domain further comprises one ormore functional signaling domains derived from at least onecostimulatory molecule as defined below. In one aspect, thecostimulatory molecule is chosen from 4-1BB (i.e., CD137) and/or CD28.In one aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen recognitiondomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a co-stimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule. In one aspect, the CAR comprises a chimeric fusion proteincomprising an extracellular antigen recognition domain, a transmembranedomain and an intracellular signaling domain comprising two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen recognition domain, wherein the leader sequence isoptionally cleaved from the antigen recognition domain (e.g., a scFv)during cellular processing and localization of the CAR to the cellularmembrane.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

The term “EGFR” refers to any mammalian mature full-length epidermalgrowth factor receptor, including human and non-human forms. The 1186amino acid human EGFR is described in Ullrich et al., Nature 309:418-425(1984)) and GenBank Accession No. AF125253 and SwissProt Acc NoP00533-2.

The term “EGFRvIII” refers to Epidermal growth factor receptor variantIII. EGFRvIII is the most common variant of EGFR observed in humantumors but is rarely observed in normal tissue. This protein resultsfrom the in-frame deletion of exons 2-7 and the generation of a novelglycine residue at the junction of exons 1 and 8 within theextra-cellular domain of the EGFR, thereby creating a tumor specificepitope. EGFRvIII is expressed in 24% to 67% of GBM, but not in normaltissues. EGFRvIII is also known as type III mutant, delta-EGFR,EGFRde2-7, and ΔEGFR and is described in U.S. Pat. Nos. 6,455,498,6,127,126, 5,981,725, 5,814,317, 5,710,010, 5,401,828, and 5,212,290.Expression of EGFRvIII may result from a chromosomal deletion, and mayalso result from aberrant alternative splicing. See Sugawa et al., 1990,Proc. Natl. Acad. Sci. 87:8602-8606.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of an intactantibody, or recombinant variants thereof, and refers to the antigenbinding domain, e.g., an antigenic determining variable region of anintact antibody, that is sufficient to confer recognition and specificbinding of the antibody fragment to a target, such as an antigen.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments, scFv antibody fragments, linearantibodies, single domain antibodies such as sdAb (either VL or VH),camelid VHH domains, and multi-specific antibodies formed from antibodyfragments. The term “scFv” refers to a fusion protein comprising atleast one antibody fragment comprising a variable region of a lightchain and at least one antibody fragment comprising a variable region ofa heavy chain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single chain polypeptide, and wherein the scFvretains the specificity of the intact antibody from which it is derived.Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The portion of the CAR composition of the invention comprising anantibody or antibody fragment thereof may exist in a variety of formswhere the antigen binding domain is expressed as part of a contiguouspolypeptide chain including, for example, a single domain antibodyfragment (sdAb), a single chain antibody (scFv) and a humanized antibody(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York; Harlow et al., 1989, In:Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to encodepolypeptides that elicit the desired immune response. Moreover, askilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample, or might bemacromolecule besides a polypeptide. Such a biological sample caninclude, but is not limited to a tissue sample, a tumor sample, a cellor a fluid with other biological components.

The term “anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in the number of metastases, an increase in life expectancy,decrease in tumor cell proliferation, decrease in tumor cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe invention in prevention of the occurrence of tumor in the firstplace.

The term “autologous” refer to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” refers to a disease characterized by the rapid anduncontrolled growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers are described herein and, include butare not limited to, glioblastoma, breast cancer, prostate cancer,ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer and the like.

The term “disease associated with expression of EGFRvIII” as used hereinincludes, but is not limited to, a disease associated with expression ofEGFRvIII or condition associated with cells which express EGFRvIIIincluding, tumor cells of various cancers such as, e.g., glioblastoma(including glioblastoma stem cells); breast, ovarian, and non-small celllung carcinomas; head and neck squamous cell carcinoma; medulloblastoma,colorectal cancer, prostate cancer, and bladder carcinoma. Without beingbound to a particular theory or mechanism, it is believed that byeliciting an antigen-specific response against EGFRvIII, the CARsdisclosed herein provide for one or more of the following: targeting anddestroying EGFRvIII-expressing tumor cells, reducing or eliminatingtumors, facilitating infiltration of immune cells to the tumor site, andenhancing/extending anti-tumor responses. Because EGFRvIII is notexpressed at detectable levels in normal (i.e., non-cancerous) tissue,it is contemplated that the inventive CARs advantageously substantiallyavoid targeting/destroying normal tissues and cells.

The term “conservative sequence modifications” is intended to refer toamino acid modifications that do not significantly affect or alter thebinding characteristics of the antibody or antibody fragment containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody or antibody fragment of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutions areones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, one or more amino acid residues within aCAR of the invention can be replaced with other amino acid residues fromthe same side chain family and the altered CAR can be tested using thefunctional assays described herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as, but notlimited to, signal transduction via the TCR/CD3 complex. Stimulation canmediate altered expression of certain molecules, such as downregulationof TGF-β, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule,” refers to a molecule expressed by a Tcell that provides the primary cytoplasmic signaling sequence(s) thatregulate primary activation of the TCR complex in a stimulatory way forat least some aspect of the T cell signaling pathway. In one aspect, theprimary signal is initiated by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, and which leads tomediation of a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. A primarycytoplasmic signaling sequence (also referred to as a “primary signalingdomain”) that acts in a stimulatory manner may contain a signaling motifwhich is known as immunoreceptor tyrosine-based activation motif orITAM. Examples of an ITAM containing primary cytoplasmic signalingsequence that is of particular use in the invention includes, but is notlimited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma,CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as“ICOS”) and CD66d. In a specific CAR of the invention, the intracellularsignaling domain in any one or more CARS of the invention comprises anintracellular signaling sequence, e.g., a primary signaling sequence ofCD3-zeta. In a specific CAR of the invention, the primary signalingsequence of CD3-zeta is the sequence provided as SEQ ID NO:17 or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In a specific CAR of the invention, theprimary signaling sequence of CD3-zeta is the sequence provided as SEQID NO:99 or the equivalent residues from a non-human species, e.g.,mouse, rodent, monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain generates a signal that promotes an immune effector function ofthe CAR containing cell, e.g., a CART cell. Examples of immune effectorfunction, e.g., in a CART cell, include cytolytic activity and helperactivity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CART, a primary intracellularsignaling domain can comprise a cytoplasmic sequence of a T cellreceptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d DAP10 and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBan Acc. No. BAG36664.1, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain that are sufficient to functionally transmit aninitial signal necessary for T cell activation. In one aspect thecytoplasmic domain of zeta comprises residues 52 through 164 of GenBankAcc. No. BAG36664.1 or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like, that are functionalorthologs thereof. In one aspect, the “zeta stimulatory domain” or a“CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:17.In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatorydomain” is the sequence provided as SEQ ID NO:99.

The term “costimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to, an MHC class I molecule, BTLA and aToll ligand receptor, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1,LFA-1 (CD11a/CD18) and 4-1BB (CD137).

A costimulatory intracellular signaling domain can be derived from theintracellular portion of a costimulatory molecule. A costimulatorymolecule can be represented in the following protein families: TNFreceptor proteins, Immunoglobulin-like proteins, cytokine receptors,integrins, signaling lymphocytic activation molecules (SLAM proteins),and activating NK cell receptors. Examples of such molecules includeCD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically bindswith CD83, and the like.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment thereof.

The term “4-1BB” refers to member of the TNFR superfamily with an aminoacid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like; and a “4-1BB costimulatory domain” is defined as amino acidresidues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like. In one aspect, the “4-1BB costimulatory domain” is thesequence provided as SEQ ID NO:16 or the equivalent residues from anon-human species, e.g., mouse, rodent, monkey, ape and the like.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” as used herein refers to the subunitsequence identity between two polymeric molecules, e.g., between twonucleic acid molecules, such as, two DNA molecules or two RNA molecules,or between two polypeptide molecules. When a subunit position in both ofthe two molecules is occupied by the same monomeric subunit; e.g., if aposition in each of two DNA molecules is occupied by adenine, then theyare homologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibodies/antibody fragment can comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

The term “human” antibody refers to fully human antibodies as well aseffectively human antibodies. “Fully human” refers to an immunoglobulin,such as an antibody or antibody fragment, where the whole molecule is ofhuman origin or consists of an amino acid sequence identical to a humanform of the antibody or immunoglobulin. An “effectively human” antibodyis an antibody that includes a sufficient number of human amino acidpositions such that the antibody does not elicit an immunogenic responsein a normal human.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive promoter” refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible promoter” refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific promoter” refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n(SEQ ID NO: 112), where n is a positive integer equal to or greaterthan 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 andn=10. In one embodiment, the flexible polypeptide linkers include, butare not limited to, (Gly4 Ser)4 (SEQ ID NO: 113) or (Gly4 Ser)3 (SEQ IDNO: 114). In another embodiment, the linkers include multiple repeats of(Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 112). Also included withinthe scope of the invention are linkers described in WO2012/138475,incorporated herein by reference).

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:115), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin's lymphoma, non-Hodgkins lymphoma, leukemias, uterinecancer, cervical cancer, bladder cancer, kidney cancer andadenocarcinomas such as breast cancer, prostate cancer, ovarian cancer,pancreatic cancer, and the like.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner (e.g., a stimulatoryand/or costimulatory molecule present on a T cell) protein present in asample, but which antibody or ligand does not substantially recognize orbind other molecules in the sample.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

Description

Provided herein are compositions of matter and methods of use for thetreatment of a disease such as cancer using an anti-EGFRvIII chimericantigen receptors (CAR).

In one aspect, the invention provides a number of chimeric antigenreceptors comprising an antibody or antibody fragment engineered tospecifically bind to an EGFRvIII protein. In one aspect, the inventionprovides a cell (e.g., T cell) engineered to express a CAR, wherein theCAR T cell (“CART”) exhibits an antitumor property. In one aspect a cellis transformed with the CAR and the CAR is expressed on the cellsurface. In some embodiments, the cell (e.g., T cell) is transduced witha viral vector encoding a CAR. In some embodiments, the viral vector isa retroviral vector. In some embodiments, the viral vector is alentiviral vector. In some such embodiments, the cell may stably expressthe CAR. In another embodiment, the cell (e.g., T cell) is transfectedwith a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some suchembodiments, the cell may transiently express the CAR.

In one aspect, the EGFRvIII protein binding portion of the CAR is a scFvantibody fragment. In one aspect such antibody fragments are functionalin that they retain the equivalent binding affinity, e.g., they bind thesame antigen with comparable efficacy, as the IgG antibody from which itis derived. In one aspect such antibody fragments are functional in thatthey provide a biological response that can include, but is not limitedto, activation of an immune response, inhibition of signal-transductionorigination from its target antigen, inhibition of kinase activity, andthe like, as will be understood by a skilled artisan.

In one aspect, the EGFRvIII antigen binding domain of the CAR is amurine scFv antibody fragment. In another aspect, the EGFRvIII antigenbinding domain of the CAR is a scFv antibody fragment that is humanizedcompared to the murine sequence of the scFv from which it is derived.Generation of an exemplary parental murine monoclonal antibody againstEGFRvIII (3C10) is disclosed in Okamoto et al. (British J. Cancer 1996,73:1366-1372). An exemplary fully human antibody against EGFRvIII (139)is disclosed in Morgan et al. (2012) Human Gene Therapy, 23: 1043-1953,incorporated herein by reference. In one aspect, the scFv for the murinesequence comprises SEQ ID NO: 11. Humanization of this mouse scFv may bedesired for the clinical setting, where the mouse-specific residues mayinduce a human-anti-mouse antigen (HAMA) response in patients whoreceive EGFRvIII treatment, e.g., treatment with T cells transduced withthe EGFRvIII construct.

In one aspect, the anti-EGFRvIII binding domain portion of a CAR isencoded by a transgene whose sequence has been codon optimized forexpression in a mammalian cell. In one aspect, entire CAR construct ofthe invention is encoded by a transgene whose entire sequence has beencodon optimized for expression in a mammalian cell. Codon optimizationrefers to the discovery that the frequency of occurrence of synonymouscodons (i.e., codons that code for the same amino acid) in coding DNA isbiased in different species. Such codon degeneracy allows an identicalpolypeptide to be encoded by a variety of nucleotide sequences. Avariety of codon optimization methods is known in the art, and include,e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and6,114,148.

In one aspect, the anti-EGFRvIII binding domain of a CAR is a humanizedanti-EGFRvIII binding domain. For example, in one embodiment, theanti-EGFRvIII binding domain comprises the scFv portion provided in SEQID NO:38. In one aspect, the humanized anti-EGFRvIII binding domaincomprises the scFv portion provided in SEQ ID NO:44. In one aspect, thehumanized anti-EGFRvIII binding domain comprises the scFv portionprovided in SEQ ID NO:50. In one aspect, the humanized anti-EGFRvIIIbinding domain comprises the scFv portion provided in SEQ ID NO:56. Inone aspect, the humanized anti-EGFRvIII binding domain comprises thescFv portion provided in SEQ ID NO:62. In one aspect, the humanizedanti-EGFRvIII binding domain comprises the scFv portion provided in SEQID NO:68. In one aspect, the humanized anti-EGFRvIII binding domaincomprises the scFv portion provided in SEQ ID NO:74. In one aspect, thehumanized anti-EGFRvIII binding domain comprises the scFv portionprovided in SEQ ID NO:80. In one aspect, the humanized anti-EGFRvIIIbinding domain comprises the scFv portion provided in SEQ ID NO:86.

In one aspect, a CAR disclosed herein includes an antigen binding domainof a specific antibody with an intracellular signaling domain. Forexample, in some aspects, the intracellular signaling domain includes,but is not limited to, CD3-zeta chain, 4-1BB and CD28 signaling modulesand combinations thereof.

In one aspect, the antigen binding domain binds to EGFRvIII. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:43. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:49. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:55. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:61. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:67. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:73. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:79. In oneaspect, the CAR comprises the sequence provided in SEQ ID NO:85.

In one aspect, CAR comprises at least one intracellular signaling domainselected from the group consisting of a CD137 (4-1BB) signaling domain,a CD28 signaling domain, a CD3zeta signal domain, and any combinationthereof. In one aspect, the CAR comprises at least one intracellularsignaling domain of one or more costimulatory molecule(s) other than aCD137 (4-1BB) or CD28, a CD3zeta signal domain, and any combinationthereof.

Furthermore, the present invention provides CAR compositions and theiruse in medicaments or methods for treating, among other diseases, canceror any malignancy or autoimmune diseases involving cells or tissueswhich express EGFRvIII.

The present invention also provides compositions and methods foroverexpression of miR-17-92, e.g., in a CAR-expressing cell, e.g., a Tcell. In one aspect, transgene-derived overexpression of miR-17-92provides a CAR-transduced T-cell with improved resistance againsttumor-induced immunosuppression and chemotherapy, thereby promotinglong-lasting therapeutic effects.

Chimeric Antigen Receptor (CAR)

The present invention encompasses a recombinant DNA construct comprisingsequences encoding a CAR, wherein the CAR comprises an antibody fragmentthat binds specifically to EGFRvIII, e.g., a human antibody fragmentthat specifically binds to EGFRvIII. In one aspect, the EGFRvIII ishuman EGFRvIII, and the nucleic acid sequence encoding the antibodyfragment is contiguous with, and in the same reading frame as a nucleicacid sequence encoding an intracellular signaling domain. Theintracellular signaling domain can comprise a costimulatory signalingdomain and/or a primary signaling domain, e.g., a zeta chain. Thecostimulatory signaling domain refers to a portion of the CAR comprisingat least a portion of the intracellular domain of a costimulatorymolecule.

In specific aspects, a CAR construct of the invention comprises a scFvdomain selected from the group consisting of SEQ ID NO:38, SEQ ID NO:44,SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74,SEQ ID NO:80, and SEQ ID NO:86, wherein the scFv may be preceded by anoptional leader sequence such as provided in SEQ ID NO: 13, and followedby an optional hinge sequence such as provided in SEQ ID NO:14 or SEQ IDNO:104 or SEQ ID NO:106 or SEQ ID NO:108, a transmembrane region such asprovided in SEQ ID NO:15, an intracellular signalling domain thatincludes SEQ ID NO:16 or SEQ ID NO:102 and a CD3 zeta sequence thatincludes SEQ ID NO:17 or SEQ ID NO:99, wherein the domains arecontiguous with and in the same reading frame to form a single fusionprotein. Also included in the invention is a nucleotide sequence thatencodes the polypeptide of each of the scFv fragments selected from thegroup consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ IDNO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQID NO: 86, and each of the domains of SEQ ID NOS: 13-17. Also includedin the invention is a nucleotide sequence that encodes the polypeptideof each of the scFv fragments selected from the group consisting of SEQID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ IDNO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ ID NO: 86, and each of thedomains of SEQ ID NOS: 13-16 and SEQ ID NO:99. In one aspect, theEGFRvIII CAR construct comprises an optional leader sequence, anextracellular antigen binding domain that specifically binds EGFRvIII, ahinge, a transmembrane domain, and an intracellular stimulatory domain.In one aspect, the EGFRvIII CAR construct comprises an optional leadersequence, an extracellular antigen binding domain that specificallybinds EGFRvIII, a hinge, a transmembrane domain, an intracellularsignaling domain that includes a costimulatory domain and a primarystimulatory domain. Specific EGFRvIII CAR constructs containing ahumanized scFv domain are provided in SEQ ID NO:43, SEQ ID NO:49, SEQ IDNO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ IDNO:85, and SEQ ID NO:90. Specific EGFRvIII CAR constructs containing amurine scFv domain is provided in SEQ ID NO:1 and SEQ ID NO:2.

An exemplary leader sequence is provided as SEQ ID NO: 13. An exemplaryhinge/spacer sequence is provided as SEQ ID NO: 14 or SEQ ID NO:104 orSEQ ID NO:106 or SEQ ID NO:108. An exemplary transmembrane domainsequence is provided as SEQ ID NO:15. An exemplary sequence of acostimulatory domain of the 4-1BB protein is provided as SEQ ID NO: 16.An exemplary sequence of a costimulatory domain of the CD27 protein isprovided as SEQ ID NO:102. An exemplary primary signaling domain of aCD3zeta domain sequence is provided as SEQ ID NO: 17. Another exemplaryprimary signaling domain of a CD3zeta domain sequence is provided as SEQID NO:99.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises the nucleic acid sequenceencoding an anti-EGFRvIII binding domain, e.g., described herein, thatis contiguous with, and in the same reading frame as a nucleic acidsequence encoding an intracellular signaling domain. In one aspect, theanti-EGFRvIII binding domain is selected from one or more of SEQ IDNO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ IDNO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ ID NO:86. In one aspect, theanti-EGFRvIII binding domain is encoded by a nucleotide sequenceprovided in a sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 57, SEQ ID NO: 63, SEQ IDNO: 69, SEQ ID NO: 75, SEQ ID NO: 81, and SEQ ID NO:98. In one aspect,the anti-EGFRvIII binding domain is encoded by SEQ ID NO: 39. In oneaspect, the anti-EGFRvIII binding domain is encoded by SEQ ID NO: 45. Inone aspect, the anti-EGFRvIII binding domain is encoded by SEQ ID NO:51. In one aspect, the anti-EGFRvIII binding domain is encoded by SEQ IDNO: 57. In one aspect, the anti-EGFRvIII binding domain is encoded bySEQ ID NO: 63. In one aspect, the anti-EGFRvIII binding domain isencoded by SEQ ID NO: 69. In one aspect, the anti-EGFRvIII bindingdomain is encoded by SEQ ID NO: 75. In one aspect, the anti-EGFRvIIIbinding domain is encoded by SEQ ID NO: 81.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises a nucleic acid sequenceencoding an anti-EGFRvIII binding domain selected from the groupconsisting of SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60,SEQ ID NO:66, SEQ ID NO:72, SEQ ID NO:78, SEQ ID NO:84, and SEQ ID NO:90wherein the sequence is contiguous with and in the same reading frame asthe nucleic acid sequence encoding an intracellular signaling domain. Anexemplary intracellular signaling domain that can be used in the CARincludes, but is not limited to, one or more intracellular signalingdomains of, e.g., CD3-zeta, CD28, 4-1BB, and the like. In someinstances, the CAR can comprise any combination of intracellularsignaling domains of CD3-zeta, CD28, 4-1BB, and the like. In one aspectthe nucleic acid construct comprises SEQ ID NO: 42. In one aspect thenucleic acid sequence of a CAR construct is SEQ ID NO:48. In one aspectthe nucleic acid construct comprises SEQ ID NO:54. In one aspect thenucleic acid construct comprises SEQ ID NO:60. In one aspect the nucleicacid construct comprises SEQ ID NO:66. In one aspect the nucleic acidconstruct comprises SEQ ID NO:72. In one aspect the nucleic acidconstruct comprises SEQ ID NO:78. In one aspect the nucleic acidconstruct comprises SEQ ID NO:84.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the nucleic acid of interest can beproduced synthetically, rather than cloned.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.

The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the nucleic acid to beexpressed, and a polyA tail, typically 50-2000 bases in length. RNA soproduced can efficiently transfect different kinds of cells. In oneembodiment, the template includes sequences for the CAR. In anembodiment, an RNA CAR vector is transduced into a T cell byelectroporation.

Antigen Binding Domain

In one aspect, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenbinding domain in a CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one aspect, the CAR-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen binding domain thatspecifically binds a desired antigen into the CAR.

In one aspect, the portion of the CAR comprising the antigen bindingdomain comprises an antigen binding domain that targets EGFRvIII. In oneaspect, the antigen binding domain targets human EGFRvIII. For example,a mouse monoclonal antibody (IgG2b) 3C10 was produced against EGFRvIIIby immunization of mice with a 14 amino acid peptide (LEEKKGNYVVTDHC;SEQ ID NO:101) including the EGFRvIII-specific fusion junction anddemonstrated highly specific recognition of EGFRvIII without anydetectable binding to wild-type EGFR (Okamoto et al, British J. Cancer1996, 73:1366-1372). Accordingly, in some embodiments, the antigenbinding domain targets an amino acid sequence, e.g., an amino acidsequence comprising an added glycine residue, within the EGFvIII fusionjunction domain. In some embodiments, the antigen binding domain targetsan one or more amino acid sequence in the amino acid sequence of SEQ IDNO:101.

The antigen binding domain can be any domain that binds to the antigenincluding, but not limited to, a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, and the like. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise human or humanized residues for the antigen binding domainof an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a humanantibody or an antibody fragment. In another aspect, the antigen bindingdomain comprises a humanized antibody or antibody fragment. In oneembodiment, the anti-EGFRvIII binding domain comprises one or more(e.g., one, two, or all three) light chain complementary determiningregion 1 (LC CDR1), light chain complementary determining region 2 (LCCDR2), and light chain complementary determining region 3 (LC CDR3) ofan anti-EGFRvIII binding domain described herein, and one or more (e.g.,one, two, or all three) heavy chain complementary determining region 1(HC CDR1), heavy chain complementary determining region 2 (HC CDR2), andheavy chain complementary determining region 3 (HC CDR3) of ananti-EGFRvIII binding domain described herein. In one embodiment, theanti-EGFRvIII binding domain comprises a light chain variable regiondescribed herein and/or a heavy chain variable region described herein.In one embodiment, the anti-EGFRvIII binding domain is a scFv comprisinga light chain variable region and a heavy chain variable region of anamino acid sequence, e.g., a light chain variable region and heavy chainvariable region described herein. In an embodiment, the anti-EGFRvIIIbinding domain (e.g., an scFv) comprises: a light chain variable regioncomprising an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a lightchain variable region provided herein, or a sequence with 85-99% (e.g.,90-99%, or 95-99%) identity to an amino acid sequence provided herein;and/or a heavy chain variable region comprising an amino acid sequencehaving at least one, two or three modifications (e.g., substitutions)but not more than 30, 20 or 10 modifications (e.g., substitutions) of anamino acid sequence of a heavy chain variable region provided herein, ora sequence with 85-99% (e.g., 90-99%, or 95-99%) identity to an aminoacid sequence provided herein. In one aspect, the antigen binding domaincomprises one or more sequence selected from the group consisting of SEQID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ IDNO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ ID NO:86. In one aspect thehumanized CAR is selected from one or more sequence selected from thegroup consisting of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ IDNO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, and SEQID NO:90.

In some aspects, a non-human antibody is humanized, where specificsequences or regions of the antibody are modified to increase similarityto an antibody naturally produced in a human or fragment thereof. In oneaspect, the antigen binding domain is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., 2002, J. Immunol., 169:1119-25;Caldas et al., 2000, Protein Eng., 13(5):353-60; Morea et al., 2000,Methods, 20:267-79; Baca et al., 1997, J. Biol. Chem., 272:10678-84;Roguska et al., 1996, Protein Eng., 9(10):895-904; Couto et al., 1995,Cancer Res., 55:5973s-5977; Couto et al., 1995, Cancer Res.,55(8):1717-22; Sandhu 1994 Gene, 150(2):409-10; and Pedersen et al.,1994, J. Mol. Biol., 235(3):959-73, each of which is incorporated hereinin its entirety by reference. Often, framework residues in the frameworkregions will be substituted with the corresponding residue from the CDRdonor antibody to alter, for example improve, antigen binding. Theseframework substitutions are identified by methods well-known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized antibodies and antibody fragments, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. Humanized antibodies areoften human antibodies in which some CDR residues and possibly someframework (FR) residues are substituted by residues from analogous sitesin rodent antibodies. Humanization of antibodies and antibody fragmentscan also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al.,PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332),the contents of which are incorporated herein by reference herein intheir entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), thecontents of which are incorporated herein by reference herein in theirentirety).

In some aspects, the portion of a CAR composition of the invention thatcomprises an antibody fragment is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies and antibody fragments are prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, e.g., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody or antibody fragment characteristic, such asincreased affinity for the target antigen, is achieved. In general, theCDR residues are directly and most substantially involved in influencingantigen binding.

In one aspect, the anti-EGFRvIII binding domain is, for example, a Fv, aFab, or a (Fab′)2, or a bi-functional (e.g. bi-specific) hybrid antibody(e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In oneaspect, an antibody fragment provided herein is a scFv. In one aspect,the scFv binds an EGFRvIII protein but not wild type EGFR. In someinstances, a human scFv may also be derived from a yeast displaylibrary.

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of an scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids, intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n (SEQ ID NO: 37), where n is a positiveinteger equal to or greater than 1. In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO: 113) or (Gly₄Ser)₃ (SEQ ID NO: 114). Variation inthe linker length may retain or enhance activity, giving rise tosuperior efficacy in activity studies.

Stability and Mutations

The stability of an anti-EGFRvIII binding domain, e.g., scFv molecules(e.g., soluble scFv), can be evaluated in reference to the biophysicalproperties (e.g., thermal stability) of a conventional control scFvmolecule or a full length antibody. In one embodiment, the humanizedscFv has a thermal stability that is greater than about 0.1, about 0.25,about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees,about 13 degrees, about 14 degrees, or about 15 degrees Celsius than acontrol binding molecule (e.g. a conventional scFv molecule) in thedescribed assays.

The improved thermal stability of the anti-EGFRvIII binding domain,e.g., scFv, is subsequently conferred to the entire EGFRvIII CARconstruct, leading to improved therapeutic properties of the EGFRvIIICAR construct. The thermal stability of the anti-EGFRvIII bindingdomain, e.g., scFv, can be improved by at least about 2° C. or 3° C. ascompared to a conventional antibody. In one embodiment, theanti-EGFRvIII binding domain, e.g., scFv, has a 1° C. improved thermalstability as compared to a conventional antibody. In another embodiment,the anti-EGFRvIII binding domain, e.g., scFv, has a 2° C. improvedthermal stability as compared to a conventional antibody. In anotherembodiment, the anti-EGFRvIII binding domain, e.g., scFv, has a 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15° C. improved thermal stability ascompared to a conventional antibody. Comparisons can be made, forexample, between the scFv molecules disclosed herein and scFv moleculesor Fab fragments of an antibody from which the scFv VH and VL werederived. Thermal stability can be measured using methods known in theart. For example, in one embodiment, Tm can be measured. Methods formeasuring Tm and other methods of determining protein stability aredescribed in more detail below.

Mutations in scFv (arising through humanization or direct mutagenesis ofthe soluble scFv) alter the stability of the scFv and improve theoverall stability of the scFv and the EGFRvIII CAR construct. Stabilityof the humanized scFv is compared against the murine scFv usingmeasurements such as Tm, temperature denaturation and temperatureaggregation. The binding capacity of the mutant scFvs can be determinedusing assays described in the Examples.

In one embodiment, the anti-EGFRvIII binding domain, e.g., scFv,comprises at least one mutation arising from the humanization processsuch that the mutated scFv confers improved stability to the EGFRvIIIconstruct. In another embodiment, the anti-EGFRvIII binding domain,e.g., scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutationsarising from the humanization process such that the mutated scFv confersimproved stability to the EGFRvIII construct.

Methods of Evaluating Protein Stability

The stability of an antigen binding domain may be assessed using, e.g.,the methods described below. Such methods allow for the determination ofmultiple thermal unfolding transitions where the least stable domaineither unfolds first or limits the overall stability threshold of amultidomain unit that unfolds cooperatively (e.g. a multidomain proteinwhich exhibits a single unfolding transition). The least stable domaincan be identified in a number of additional ways. Mutagenesis can beperformed to probe which domain limits the overall stability.Additionally, protease resistance of a multidomain protein can beperformed under conditions where the least stable domain is known to beintrinsically unfolded via DSC or other spectroscopic methods (Fontana,et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol.393: 672-692). Once the least stable domain is identified, the sequenceencoding this domain (or a portion thereof) may be employed as a testsequence in the methods.

a) Thermal Stability

The thermal stability of the compositions may be analyzed using a numberof non-limiting biophysical or biochemical techniques known in the art.In certain embodiments, thermal stability is evaluated by analyticalspectroscopy.

An exemplary analytical spectroscopy method is Differential Scanningcalorimetry (DSC). DSC employs a calorimeter which is sensitive to theheat absorbances that accompany the unfolding of most proteins orprotein domains (see, e.g. Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, asample of the protein is inserted into the calorimeter and thetemperature is raised until the Fab or scFv unfolds. The temperature atwhich the protein unfolds is indicative of overall protein stability.

Another exemplary analytical spectroscopy method is Circular Dichroism(CD) spectroscopy. CD spectrometry measures the optical activity of acomposition as a function of increasing temperature. Circular dichroism(CD) spectroscopy measures differences in the absorption of left-handedpolarized light versus right-handed polarized light which arise due tostructural asymmetry. A disordered or unfolded structure results in a CDspectrum very different from that of an ordered or folded structure. TheCD spectrum reflects the sensitivity of the proteins to the denaturingeffects of increasing temperature and is therefore indicative of aprotein's thermal stability (see van Mierlo and Steemsma, J.Biotechnol., 79(3):281-98, 2000).

Another exemplary analytical spectroscopy method for measuring thermalstability is Fluorescence Emission Spectroscopy (see van Mierlo andSteemsma, supra). Yet another exemplary analytical spectroscopy methodfor measuring thermal stability is Nuclear Magnetic Resonance (NMR)spectroscopy (see, e.g. van Mierlo and Steemsma, supra).

The thermal stability of a composition can be measured biochemically. Anexemplary biochemical method for assessing thermal stability is athermal challenge assay. In a “thermal challenge assay”, a compositionis subjected to a range of elevated temperatures for a set period oftime. For example, in one embodiment, test scFv molecules or moleculescomprising scFv molecules are subject to a range of increasingtemperatures, e.g., for 1-1.5 hours. The activity of the protein is thenassayed by a relevant biochemical assay. For example, if the protein isa binding protein (e.g. an scFv or scFv-containing polypeptide) thebinding activity of the binding protein may be determined by afunctional or quantitative ELISA.

Such an assay may be done in a high-throughput format and thosedisclosed in the Examples using E. coli and high throughput screening. Alibrary of anti-EGFRvIII binding domain, e.g., scFv, variants may becreated using methods known in the art. Anti-EGFRvIII binding domain,e.g., scFv, expression may be induced and the anti-EGFRvIII bindingdomain, e.g., scFv, may be subjected to thermal challenge. Thechallenged test samples may be assayed for binding and thoseanti-EGFRvIII binding domains, e.g., scFvs, which are stable may bescaled up and further characterized.

Thermal stability is evaluated by measuring the melting temperature (Tm)of a composition using any of the above techniques (e.g. analyticalspectroscopy techniques). The melting temperature is the temperature atthe midpoint of a thermal transition curve wherein 50% of molecules of acomposition are in a folded state (See e.g., Dimasi et al. (2009) J. MolBiol. 393: 672-692). In one embodiment, Tm values for an anti-EGFRvIIIbinding domain, e.g., scFv, are about 40° C., 41° C., 42° C., 43° C.,44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C.,53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C.,62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C.,71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C.,80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C.,89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C.,98° C., 99° C., 100° C. In one embodiment, Tm values for an IgG is about40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C.,76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C.,85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C.,94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C. In oneembodiment, Tm values for an multivalent antibody is about 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77°C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86°C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95°C., 96° C., 97° C., 98° C., 99° C., 100° C.

Thermal stability is also evaluated by measuring the specific heat orheat capacity (Cp) of a composition using an analytical calorimetrictechnique (e.g. DSC). The specific heat of a composition is the energy(e.g. in kcal/mol) is required to rise by 1° C., the temperature of 1mol of water. As large Cp is a hallmark of a denatured or inactiveprotein composition. The change in heat capacity (ΔCp) of a compositionis measured by determining the specific heat of a composition before andafter its thermal transition. Thermal stability may also be evaluated bymeasuring or determining other parameters of thermodynamic stabilityincluding Gibbs free energy of unfolding (ΔG), enthalpy of unfolding(ΔH), or entropy of unfolding (ΔS). One or more of the above biochemicalassays (e.g. a thermal challenge assay) are used to determine thetemperature (i.e. the T_(C) value) at which 50% of the compositionretains its activity (e.g. binding activity).

In addition, mutations to the anti-EGFRvIII binding domain, e.g., scFv,alter the thermal stability of the anti-EGFRvIII binding domain, e.g.,scFv, compared with the unmutated anti-EGFRvIII binding domain, e.g.,scFv. When the humanized anti-EGFRvIII binding domain, e.g., scFv, isincorporated into an anti-EGFRvIII CAR construct, the anti-EGFRvIIIbinding domain, e.g., humanized scFv confers thermal stability to theoverall anti-EGFRvIII CAR construct. In one embodiment, theanti-EGFRvIII binding domain, e.g., scFv, comprises a single mutationthat confers thermal stability to the anti-EGFRvIII binding domain,e.g., scFv. In another embodiment, the anti-EGFRvIII binding domain,e.g., scFv, comprises multiple mutations that confer thermal stabilityto the anti-EGFRvIII binding domain, e.g., scFv. In one embodiment, themultiple mutations in the anti-EGFRvIII binding domain, e.g., scFv, havean additive effect on thermal stability of the anti-EGFRvIII bindingdomain, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by measuring itspropensity to aggregate. Aggregation can be measured by a number ofnon-limiting biochemical or biophysical techniques. For example, theaggregation of a composition may be evaluated using chromatography, e.g.Size-Exclusion Chromatography (SEC). SEC separates molecules on thebasis of size. A column is filled with semi-solid beads of a polymericgel that will admit ions and small molecules into their interior but notlarge ones. When a protein composition is applied to the top of thecolumn, the compact folded proteins (i.e. non-aggregated proteins) aredistributed through a larger volume of solvent than is available to thelarge protein aggregates. Consequently, the large aggregates move morerapidly through the column, and in this way the mixture can be separatedor fractionated into its components. Each fraction can be separatelyquantified (e.g. by light scattering) as it elutes from the gel.Accordingly, the % aggregation of a composition can be determined bycomparing the concentration of a fraction with the total concentrationof protein applied to the gel. Stable compositions elute from the columnas essentially a single fraction and appear as essentially a single peakin the elution profile or chromatogram.

c) Binding Affinity

The stability of a composition can be assessed by determining its targetbinding affinity. A wide variety of methods for determining bindingaffinity are known in the art. An exemplary method for determiningbinding affinity employs surface plasmon resonance. Surface plasmonresonance is an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627;Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson,B., et al. (1991) Anal. Biochem. 198:268-277.

In one aspect, the antigen binding domain of the CAR comprises an aminoacid sequence that is homologous to an antigen binding domain amino acidsequence described herein, and the antigen binding domain retains thedesired functional properties of the anti-EGFRvIII antibody fragmentsdescribed herein. In one specific aspect, the CAR composition of theinvention comprises an antibody fragment. In a further aspect, thatantibody fragment comprises an scFv.

In various aspects, the antigen binding domain of the CAR is engineeredby modifying one or more amino acids within one or both variable regions(e.g., VH and/or VL), for example within one or more CDR regions and/orwithin one or more framework regions. In one specific aspect, the CARcomposition of the invention comprises an antibody fragment. In afurther aspect, that antibody fragment comprises an scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, e.g., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM 120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the VH or VL ofan anti-EGFRvIII binding domain, e.g., scFv, comprised in the CAR can bemodified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VHor VL framework region of the anti-EGFRvIII binding domain, e.g., scFv.The present invention contemplates modifications of the entire CARconstruct, e.g., modifications in one or more amino acid sequences ofthe various domains of the CAR construct in order to generatefunctionally equivalent molecules. The CAR construct can be modified toretain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity of the starting CAR construct.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR is used.In some instances, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membraneproteins, e.g., to minimize interactions with other members of thereceptor complex. In one aspect, the transmembrane domain is capable ofhomodimerization with another CAR on the CART cell surface. In adifferent aspect, the amino acid sequence of the transmembrane domainmay be modified or substituted so as to minimize interactions with thebinding domains of the native binding partner present in the same CART.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge,e.g., an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge orspacer comprises (e.g., consists of) the amino acid sequence of SEQ IDNO:14. In one aspect, the transmembrane domain comprises (e.g., consistsof) a transmembrane domain of SEQ ID NO: 15.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequenceESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ IDNO:104). In some embodiments, the hinge or spacer comprises a hingeencoded by a nucleotide sequence ofGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:105).

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:106). Insome embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence ofAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACG TGACTGACCATT(SEQ ID NO:107).

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect, a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, e.g., between 2 and 10amino acids in length, may form the linkage between the transmembranedomain and the cytoplasmic region of the CAR. A glycine-serine doubletis an example of a suitable linker. For example, in one aspect, thelinker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:108).In some embodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:109).

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR includes an intracellularsignaling domain. An intracellular signaling domain is generallyresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been introduced. Theterm “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic signaling domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, and CD66d. In one embodiment, a CAR of the invention, e.g., a CARselected from the group consisting of SEQ ID NO:43, SEQ ID NO:49, SEQ IDNO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, and SEQID NO:85, comprises a intracellular signaling domain, e.g., a primarysignaling domain, of CD3-zeta. In one embodiment, a primary signalingdomain comprises a modified ITAM domain, e.g., a mutated ITAM domainwhich has altered (e.g., increased or decreased) activity as compared tothe native ITAM domain. In one embodiment, a primary signaling domaincomprises a modified ITAM-containing primary intracellular signalingdomain, e.g., an optimized and/or truncated ITAM-containing primaryintracellular signaling domain. In an embodiment, a primary signalingdomain comprises one, two, three, four or more ITAM motifs.

The intracellular signaling domain of the CAR can comprise the CD3-zetasignaling domain by itself or it can be combined with any other desiredintracellular signaling domain(s) useful in the context of a CAR of theinvention. For example, the intracellular signaling domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Forexample, CD27 costimulation has been demonstrated to enhance expansion,effector function, and survival of human CART cells in vitro andaugments human T cell persistence and antitumor activity in vivo (Songet al. Blood. 2012; 119(3):696-706).

The intracellular signaling sequences within the cytoplasmic portion ofa CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequence. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signalling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 16. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 17.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQID NO:102). In one aspect, the signalling domain of CD27 is encoded by anucleic acid sequence ofAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCC(SEQ ID NO:103).

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (EGFRvIII) or adifferent target.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells. In some embodiments, thepopulation of CAR-expressing cells comprises a mixture of cellsexpressing different CARs. For example, in one embodiment, thepopulation of CART cells can include a first cell expressing a CARhaving an anti-EGFRvIII binding domain described herein, and a secondcell expressing a CAR having a different anti-EGFRvIII binding domain,e.g., an anti-EGFRvIII binding domain described herein that differs fromthe anti-EGFRvIII binding domain in the CAR expressed by the first cell.As another example, the population of CAR-expressing cells can include afirst cell expressing a CAR that includes an anti-EGFRvIII bindingdomain, e.g., as described herein, and a second cell expressing a CARthat includes an antigen binding domain to a target other than EGFRvIII.In one embodiment, the population of CAR-expressing cells includes,e.g., a first cell expressing a CAR that includes a primaryintracellular signaling domain, and a second cell expressing a CAR thatincludes a secondary signaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having ananti-EGFRvIII domain described herein, and a second cell expressinganother agent, e.g., an agent which enhances the activity of aCAR-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,PD1, can, in some embodiments, decrease the ability of a CAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4 and TGFR beta.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. The present invention also includes a CAR encoding RNA constructthat can be directly transfected into a cell. A method for generatingmRNA for use in transfection can involve in vitro transcription (IVT) ofa template with specially designed primers, followed by polyA addition,to produce a construct containing 3′ and 5′ untranslated sequence(“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), thenucleic acid to be expressed, and a polyA tail, typically 50-2000 basesin length (SEQ ID NO: 116). RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect the EGFRvIII CAR is encoded by a messenger RNA (mRNA). Inone aspect the mRNA encoding the EGFRvIII CAR is introduced into a Tcell for production of a CART cell.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR of the present invention. For example, thetemplate for the RNA CAR comprises an extracellular region comprising asingle chain variable domain of an anti-tumor antibody; a hinge region,a transmembrane domain (e.g., a transmembrane domain of CD8a); and acytoplasmic region that includes an intracellular signaling domain,e.g., comprising the signaling domain of CD3-zeta and the signalingdomain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 117) (size can be 50-5000 T (SEQ ID NO: 118)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 119).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 120) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3′ end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Nucleic Acid Constructs Encoding a CAR

The present invention provides nucleic acid molecules encoding one ormore CAR constructs described herein. In one aspect, the nucleic acidmolecule is provided as a messenger RNA transcript. In one aspect, thenucleic acid molecule is provided as a DNA construct.

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR),wherein the CAR comprises a anti-EGFRvIII binding domain (e.g., ahumanized anti-EGFRvIII binding domain), a transmembrane domain, and anintracellular signaling domain comprising a stimulatory domain, e.g., acostimulatory signaling domain and/or a primary signaling domain, e.g.,zeta chain. In one embodiment, the anti-EGFRvIII binding domain is ananti-EGFRvIII binding domain described herein, e.g., an anti-EGFRvIIIbinding domain which comprises a sequence selected from a groupconsisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56,SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, and SEQ ID NO:80, or asequence with 95-99% identify thereof. In one embodiment, the isolatednucleic acid molecule further comprises a sequence encoding acostimulatory domain. In one embodiment, the costimulatory domain is afunctional signaling domain of a protein selected from the groupconsisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS(CD278), and 4-1BB (CD137). In one embodiment, the costimulatory domaincomprises a sequence of SEQ ID NO:16, or a sequence with 95-99% identitythereof. In one embodiment, the transmembrane domain is transmembranedomain of a protein selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137and CD154. In one embodiment, the transmembrane domain comprises asequence of SEQ ID NO: 15, or a sequence with 95-99% identity thereof.In one embodiment, the intracellular signaling domain comprises afunctional signaling domain of 4-1BB and a functional signaling domainof CD3 zeta. In one embodiment, the intracellular signaling domaincomprises the sequence of SEQ ID NO: 16 or SEQ ID NO:102, or a sequencewith 95-99% identity thereof, and the sequence of SEQ ID NO: 17 or SEQID NO:99, or a sequence with 95-99% identity thereof, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain. In one embodiment, theanti-EGFRvIII binding domain is connected to the transmembrane domain bya hinge region, e.g., a hinge described herein. In one embodiment, thehinge region comprises SEQ ID NO:14 or SEQ ID NO:104 or SEQ ID NO:106 orSEQ ID NO:108, or a sequence with 95-99% identity thereof.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence of SEQ IDNO: 13, a scFv domain having a sequence selected from the groupconsisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56,SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80, and SEQ ID NO:86(or a sequence with 95-99% identify thereof), a hinge region of SEQ IDNO:14 or SEQ ID NO:104 or SEQ ID NO:106 or SEQ ID NO:108 (or a sequencewith 95-99% identity thereof), a transmembrane domain having a sequenceof SEQ ID NO: 15 (or a sequence with 95-99% identity thereof), a 4-1BBcostimulatory domain having a sequence of SEQ ID NO:16 or a CD27costimulatory domain having a sequence of SEQ ID NO:102 (or a sequencewith 95-99% identity thereof), and a CD3 zeta stimulatory domain havinga sequence of SEQ ID NO:17 or SEQ ID NO:99 (or a sequence with 95-99%identity thereof).

In another aspect, the invention pertains to an isolated polypeptidemolecule encoded by the nucleic acid molecule. In one embodiment, theisolated polypeptide molecule comprises a sequence selected from thegroup consisting of SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ IDNO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, and SEQID NO:90 or a sequence with 95-99% identify thereof. In one embodiment,the isolated polypeptide comprises a sequence of SEQ ID NO:73, or asequence with 95-99% identify thereof. In one embodiment, the isolatedpolypeptide comprises a sequence of SEQ ID NO:79, or a sequence with95-99% identify thereof.

In another aspect, the invention pertains to a nucleic acid moleculeencoding a chimeric antigen receptor (CAR) molecule that comprises ananti-EGFRvIII binding domain, a transmembrane domain, and anintracellular signaling domain comprising a stimulatory domain, andwherein said anti-EGFRvIII binding domain comprises a sequence selectedfrom the group consisting of SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50,SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:80,and SEQ ID NO:86, or a sequence with 95-99% identify thereof.

In one embodiment, the encoded CAR molecule further comprises a sequenceencoding a costimulatory domain. In one embodiment, the costimulatorydomain is a functional signaling domain of a protein selected from thegroup consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18)and 4-1BB (CD137). In one embodiment, the costimulatory domain comprisesa sequence of SEQ ID NO:16. In one embodiment, the transmembrane domainis a transmembrane domain of a protein selected from the groupconsisting of the alpha, beta or zeta chain of the T-cell receptor,CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137 and CD 154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO:15. In oneembodiment, the intracellular signaling domain comprises a functionalsignaling domain of 4-1BB and a functional signaling domain of zeta. Inone embodiment, the intracellular signaling domain comprises thesequence of SEQ ID NO: 16 and the sequence of SEQ ID NO: 17, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain. In one embodiment, theanti-EGFRvIII binding domain is connected to the transmembrane domain bya hinge region. In one embodiment, the hinge region comprises SEQ IDNO:14. In one embodiment, the hinge region comprises SEQ ID NO:104 orSEQ ID NO:106 or SEQ ID NO:108

In another aspect, the invention pertains to an encoded CAR moleculecomprising a leader sequence of SEQ ID NO: 13, a scFv domain having asequence selected from the group consisting of SEQ ID NO:38, SEQ IDNO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, SEQ IDNO:74, SEQ ID NO:80, and SEQ ID NO:86, or a sequence with 95-99%identify thereof, a hinge region of SEQ ID NO:14 or SEQ ID NO:104 or SEQID NO:106 or SEQ ID NO:108, a transmembrane domain having a sequence ofSEQ ID NO: 15, a 4-1BB costimulatory domain having a sequence of SEQ IDNO:16 or a CD27 costimulatory domain having a sequence of SEQ ID NO:102,and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 orSEQ ID NO:99. In one embodiment, the encoded CAR molecule comprises asequence selected from a group consisting of SEQ ID NO:43, SEQ ID NO:49,SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79,SEQ ID NO:85, and SEQ ID NO:90, or a sequence with 95-99% identifythereof. In one embodiment, the encoded CAR molecule comprises asequence of SEQ ID NO:73, or a sequence with 95-99% identify thereof. Inone embodiment, the isolated CAR molecule comprises a sequence of SEQ IDNO:79, or a sequence with 95-99% identify thereof.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a nanoparticle, e.g., a liposome or other suitablesub-micron sized delivery system. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a CARencoding nucleic acid molecule. In one aspect, a CAR vector can bedirectly transduced into a cell, e.g., a T cell. In one aspect, thevector is a cloning or expression vector, e.g., a vector including, butnot limited to, one or more plasmids (e.g., expression plasmids, cloningvectors, minicircles, minivectors, double minute chromosomes),retroviral and lentiviral vector constructs. In one aspect, the vectoris capable of expressing the CAR construct in mammalian T cells. In oneaspect, the mammalian T cell is a human T cell.

Sources of T Cells

Prior to expansion and genetic modification, a source of T cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Incertain aspects of the present invention, any number of T cell linesavailable in the art, may be used. In certain aspects of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred aspect, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one aspect of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative aspect, the wash solution lackscalcium and may lack magnesium or may lack many if not all divalentcations. Initial activation steps in the absence of calcium can lead tomagnified activation. As those of ordinary skill in the art wouldreadily appreciate a washing step may be accomplished by methods knownto those in the art, such as by using a semi-automated “flow-through”centrifuge (for example, the Cobe 2991 cell processor, the BaxterCytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca-free,Mg-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample may be removed and the cells directly resuspended in culturemedia.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. A specific subpopulation of T cells, such as CD3+, CD28+,CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated bypositive or negative selection techniques. For example, in one aspect, Tcells are isolated by incubation with anti-CD3/anti-CD28 (e.g.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T cells. In oneaspect, the time period is about 30 minutes. In a further aspect, thetime period ranges from 30 minutes to 36 hours or longer and all integervalues there between. In a further aspect, the time period is at least1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the timeperiod is 10 to 24 hours. In one aspect, the incubation time period is24 hours. Longer incubation times may be used to isolate T cells in anysituation where there are few T cells as compared to other cell types,such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissueor from immunocompromised individuals. Further, use of longer incubationtimes can increase the efficiency of capture of CD8+ T cells. Thus, bysimply shortening or lengthening the time T cells are allowed to bind tothe CD3/CD28 beads and/or by increasing or decreasing the ratio of beadsto T cells (as described further herein), subpopulations of T cells canbe preferentially selected for or against at culture initiation or atother time points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain aspects, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certainaspects, T regulatory cells are depleted by anti-C25 conjugated beads orother similar method of selection.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 2 billion cells/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In a further aspect, greater than 100 millioncells/ml is used. In a further aspect, a concentration of cells of 10,15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet oneaspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 millioncells/ml is used. In further aspects, concentrations of 125 or 150million cells/ml can be used. Using high concentrations can result inincreased cell yield, cell activation, and cell expansion. Further, useof high cell concentrations allows more efficient capture of cells thatmay weakly express target antigens of interest, such as CD28-negative Tcells, or from samples where there are many tumor cells present (e.g.,leukemic blood, tumor tissue, etc.). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+ Tcells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one aspect a blood sample or an apheresis is taken from a generallyhealthy subject. In certain aspects, a blood sample or an apheresis istaken from a generally healthy subject who is at risk of developing adisease, but who has not yet developed a disease, and the cells ofinterest are isolated and frozen for later use. In certain aspects, theT cells may be expanded, frozen, and used at a later time. In certainaspects, samples are collected from a patient shortly after diagnosis ofa particular disease as described herein but prior to any treatments. Ina further aspect, the cells are isolated from a blood sample or anapheresis from a subject prior to any number of relevant treatmentmodalities, including but not limited to treatment with agents such asnatalizumab, efalizumab, antiviral agents, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

Activation and Expansion of T Cells

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle:cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example,10^4 to 10^9 T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28T paramagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 2 billion cells/ml is used. In one aspect, greater than 100million cells/ml is used. In a further aspect, a concentration of cellsof 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. Inyet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100million cells/ml is used. In further aspects, concentrations of 125 or150 million cells/ml can be used. Using high concentrations can resultin increased cell yield, cell activation, and cell expansion. Further,use of high cell concentrations allows more efficient capture of cellsthat may weakly express target antigens of interest, such asCD28-negative T cells. Such populations of cells may have therapeuticvalue and would be desirable to obtain in certain aspects. For example,using high concentration of cells allows more efficient selection ofCD8+ T cells that normally have weaker CD28 expression.

In one aspect of the present invention, the mixture may be cultured forseveral hours (about 3 hours) to about 14 days or any hourly integervalue in between. In one aspect, the mixture may be cultured for 21days. In one aspect of the invention the beads and the T cells arecultured together for about eight days. In one aspect, the beads and Tcells are cultured together for 2-3 days. Several cycles of stimulationmay also be desired such that culture time of T cells can be 60 days ormore. Conditions appropriate for T cell culture include an appropriatemedia (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a EGFRvIII CAR is constructed, various assays can be used toevaluate the activity of the molecule, such as but not limited to, theability to expand T cells following antigen stimulation, sustain T cellexpansion in the absence of re-stimulation, and anti-cancer activitiesin appropriate in vitro and animal models. Assays to evaluate theeffects of a EGFRvIII CAR are described in further detail below

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4⁺ and CD8⁺ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-ζ cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-ζ chain. The same T cell subsets are usedfor SDS-PAGE analysis under non-reducing conditions to permit evaluationof covalent dimer formation.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1a,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either EGFRvIII⁺ U-87 cells(U-87-EGFRvIII), wild-type U-87 cells (U-87 wild type) or K562 cellsexpressing hCD32 and 4-1BBL in the presence of antiCD3 and anti-CD28antibody (K562-BBL-3/28) following washing. Exogenous IL-2 is added tothe cultures every other day at 100 IU/ml. GFP⁺ T cells are enumeratedby flow cytometry using bead-based counting. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter followingstimulation with αCD3/αCD28 coated magnetic beads on day 0, andtransduction with the indicated CAR on day 1.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with targetcells, such asU87MG, BHK or CHO cells expressing EGFRvIII or EGFRwildtype (wt) or CD32 and CD137 (KT32-BBL) for a final T-cell:targetcell ratio of 1:1. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3)monoclonal antibodies are added to cultures with KT32-BBL cells to serveas a positive control for stimulating T-cell proliferation since thesesignals support long-term CD8⁺ T cell expansion ex vivo. T cells areenumerated in cultures using CountBright™ fluorescent beads (Invitrogen,Carlsbad, Calif.) and flow cytometry as described by the manufacturer.CAR⁺ T cells are identified by GFP expression using T cells that areengineered with eGFP-2A linked CAR-expressing lentiviral vectors. ForCAR+ T cells not expressing GFP, the CAR+ T cells are detected withbiotinylated recombinant EGFRvIII protein and a secondary avidin-PEconjugate. CD4+ and CD8⁺ expression on T cells are also simultaneouslydetected with specific monoclonal antibodies (BD Biosciences). Cytokinemeasurements are performed on supernatants collected 24 hours followingre-stimulation using the human TH1/TH2 cytokine cytometric bead arraykit (BD Biosciences, San Diego, Calif.) according the manufacturer'sinstructions. Fluorescence is assessed using a FACScalibur flowcytometer, and data is analyzed according to the manufacturer'sinstructions.

Cytotoxicity can be assessed by a standard 51Cr-release assay. See,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly,target cells (U87MG, BHK or CHO cells expressing EGFRvIII or EGFRwildtype (wt) are loaded with 51Cr (as NaCrO4, New England Nuclear,Boston, Mass.) at 37° C. for 2 hours with frequent agitation, washedtwice in complete RPMI and plated into microtiter plates. Effector Tcells are mixed with target cells in the wells in complete RPMI atvarying ratios of effector cell:target cell (E:T). Additional wellscontaining media only (spontaneous release, SR) or a 1% solution oftriton-X 100 detergent (total release, TR) are also prepared. After 4hours of incubation at 37° C., supernatant from each well is harvested.Released ⁵¹Cr is then measured using a gamma particle counter (PackardInstrument Co., Waltham, Mass.). Each condition is performed in at leasttriplicate, and the percentage of lysis is calculated using the formula:% Lysis=(ER−SR)/(TR−SR), where ER represents the average 51Cr releasedfor each experimental condition. Alternative cytotoxicity assays mayalso be used, such as flow based cytotoxicity assays, as described inExample 8.

Click beetle red and click beetle green luciferase can be used tosimultaneously follow tumor progression and T cell trafficking, as eachuse the same luciferin substrate but emit light at the opposite ends ofthe visible light spectrum.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theEGFRvIII CAR constructs of the invention.

Therapeutic Application for EGFRvIII Expressing Diseases and Disorders

EGFRvIII is a tumor specific, ligand-independent, constitutively activevariant of the epidermal growth factor receptor. The present inventionprovides compositions and methods for treating diseases and disordersassociated with EGFRvIII. An example of a disease or disorder associatedwith EGFRvIII is glioma.

Glioma refers to a cancer of the central nervous system that begins inglial cells (e.g., cells that surround and support nerve cells andincludes oligodendrocytes, astrocytes, microglia, and ependymal cells).Gliomas are particularly serious in terms of both incidence andmalignancy, and are classified into seven or more types such asglioblastoma and anaplastic astrocytoma according to their detailedpathological tissue type. Disease stage (tumor size, presence of distalmetastasis) and histological malignancy are used when determining thedegree of malignancy of primary brain tumors. Histological malignancy isclassified into four levels, i.e., G1 to G4 according to the Guidelinesfor the Treatment of Brain Tumors ((2002) Kanehara & Co., Ltd.), andthese correspond to WHO1 to WHO4, respectively. The larger the number,the higher the degree of malignancy. For example, the malignancy ofglioblastoma is G4 (WHO4), while the malignancy of anaplasticastrocytoma is G3 (WHO3), and both G3 and G4 are classified asmalignant. Thus, according to some embodiments, the methods of thisinvention target malignant gliomas. In other aspects the inventiontargets glioblastoma multiforme (GBM). In further embodiments, thecompositions and methods of the present invention may be used in thetreatment of other gliomas including, but not limited to, anaplasticastrocytoma, giant cell glioblastoma, gliosarcoma, anaplasticoligodendroglioma, anaplastic ependymoma, choroid plexus carcinoma,anaplastic ganglioglioma, pineoblastoma, medulloepithelioma,ependymoblastoma, medulloblastoma, supratentorial primitiveneuroectodermal tumor, and atypical teratoid/rhabdoid tumor.

Glioblastoma is the most common primary brain tumor in adults. More thanhalf of the 18,000 patients diagnosed with malignant primary braintumors in US each year have glioblastoma multiforme. Glioblastomamultiforme is an anaplastic, highly cellular tumor, with highproliferation indices, microvascular proliferation and focal necrosis.Signs and symptoms depend on several factors (size, rate of growth,localization of the tumor within the brain) and are mainly representedby headache, seizures, neurological deficits, changes in mental status.Glioblastoma multiforme prognosis remains dismal. Survival time is lessthan 2 years for the majority of patients. Karnofsky performance status(KPS) is one of the most important prognostic factors: patients withKPS>70 are alive at 18 months in approx 18% of cases, compared with 13%of patients with lower KPS scores. Primary glioblastoma multiformedevelops de novo from glial cells, typically has a clinical history ofless than six months, is more common in older patients and presentssmall-cell histology. Secondary glioblastoma multiforme develops overmonths or years from pre-existing low-grade astrocytomas, predominantlyaffects younger people and presents giant-cell histology.

Malignant gliomas are also known as high grade gliomas. They can affectthe brain and the spinal cord. In some aspects, compositions and methodsof the present invention may be used to treat subjects carrying a brainmalignant glioma, for example, one that is chosen among anaplasticastrocytoma (AA), glioblastoma multiform (GBM), anaplasticoligodendroglioma (AO) and anaplastic oligoastrocytoma (AOA). In someaspects, compositions and methods of the present invention may be usedto treat subjects carrying a glioblastoma multiforme (GBM).

Glioblastoma multiforme is the most malignant stage of astrocytoma, withsurvival times of less than 2 years for most patients. Histologically,these tumors are characterized by high proliferation indices,endothelial proliferation and focal necrosis. The highly proliferativenature of these lesions likely results from multiple mitogenic effects.One of the hallmarks of GBM is endothelial proliferation. A host ofangiogenic growth factors and their receptors are found in GBMs.

There are biologic subsets of astrocytomas, which may reflect theclinical heterogeneity observed in these tumors. These subsets includebrain stem gliomas, which are a form of pediatric diffuse, fibrillaryastrocytoma that often follow a malignant course. Brain stem GBMs sharegenetic features with those adult GBMs that affect younger patients.Pleiomorphic xanthoastrocytoma (PXA) is a superficial, low-gradeastrocytic tumor that predominantly affects young adults. While thesetumors have a bizarre histological appearance, they are typicallyslow-growing tumors that may be amenable to surgical cure. Some PXAs,however, may recur as GBM. Pilocytic astrocytoma is the most commonastrocytic tumor of childhood and differs clinically andhistopathologically from the diffuse, fibrillary astrocytoma thataffects adults. Pilocytic astrocytomas do not have the same genomicalterations as diffuse, fibrillary astrocytomas. Subependymal giant cellastrocytomas (SEGA) are periventricular, low-grade astrocytic tumorsthat are usually associated with tuberous sclerosis (TS), and arehistologically identical to the so-called “candle-gutterings” that linethe ventricles of TS patients. Similar to the other tumorous lesions inTS, these are slowly-growing and may be more akin to hamartomas thantrue neoplasms. Desmoplastic cerebral astrocytoma of infancy (DCAI) anddesmoplastic infantile ganglioglioma (DIGG) are large, superficial,usually cystic, benign astrocytomas that affect children in the firstyear or two of life.

Oligodendrogliomas and oligoastrocytomas (mixed gliomas) are diffuse,primarily CNS glial tumors that are clinically and biologically mostclosely related to the diffuse, fibrillary astrocytomas. The tumors,however, are far less common than astrocytomas and have generally betterprognoses than the diffuse astrocytomas. Oligodendrogliomas andoligoastrocytomas may progress, either to WHO grade III anaplasticoligodendroglioma or anaplastic oligoastrocytoma, or to WHO grade IVGBM. Thus, the genetic changes that lead to oligodendroglial tumorsconstitute yet another pathway to GBM.

Ependymomas are a clinically diverse group of gliomas that vary fromaggressive intraventricular tumors of children to benign spinal cordtumors in adults. Transitions of ependymoma to GBM are rare. Choroidplexus tumors are also a varied group of tumors that preferentiallyoccur in the ventricular system, ranging from aggressive supratentorialintraventricular tumors of children to benign cerebellopontine angletumors of adults. Choroid plexus tumors have been reported occasionallyin patients with Li-Fraumeni syndrome and von Hippel-Lindau (VHL)disease.

Medulloblastomas are malignant, primitive tumors that arise in theposterior fossa, primarily in children. These tumors also occur in youngadults. Medulloblastomas often are surgically resected with subsequenttreatment with chemotherapy and/or radiation. They may recur locally oroccasionally as drop metastasis from the posterior fossa to the spine.Meningiomas are common intracranial tumors that arise in the meningesand compress the underlying brain. Although typically considered benignand only rarely frankly malignant, management of these tumors oftenposes clinical challenges. Histological grades of meningiomas vary withthe majority benign, WHO grade I/IV (82%); less commonly atypical, WHOII/IV (15%); and infrequently they occur as anaplastic or malignant, WHOgrade III/IV (3%).

Schwannomas are benign tumors that arise on peripheral nerves.Schwannomas may arise on cranial nerves, particularly the vestibularportion of the eighth cranial nerve (vestibular schwannomas, acousticneuromas) where they present as cerebellopontine angle masses.Hemangioblastomas are tumors of uncertain origin that are composed ofendothelial cells, pericytes and so-called stromal cells. These benigntumors most frequently occur in the cerebellum and spinal cord of youngadults. Multiple hemangioblastomas are characteristic of vonHippel-Lindau disease (VHL). Hemangiopericytomas (HPCs) are dural tumorswhich may display locally aggressive behavior and may metastasize. Thehistogenesis of dural-based hemangiopericytoma (HPC) has long beendebated, with some authors classifying it as a distinct entity andothers classifying it as a subtype of meningioma.

The symptoms of both primary and metastatic brain tumors often depend onthe location in the brain and the size of the tumor. Since variousregions of the brain are responsible for specific functions, clinicalsymptoms will vary a great deal. Tumors in the frontal lobe of the brainmay cause weakness and paralysis, mood disturbances, difficultythinking, confusion and disorientation, and wide emotional mood swings.Parietal lobe tumors may cause seizures, numbness or paralysis,difficulty with handwriting, inability to perform simple mathematicalproblems, difficulty with certain movements, and loss of the sense oftouch. Tumors in the occipital lobe can cause loss of vision in half ofeach visual field, visual hallucinations, and seizures. Temporal lobetumors can cause seizures, perceptual and spatial disturbances, andreceptive aphasia. If a tumor occurs in the cerebellum, the person mayhave ataxia, loss of coordination, headaches, and vomiting. Tumors inthe hypothalamus may cause emotional changes, and changes in theperception of hot and cold. In addition, hypothalamic tumors may affectgrowth and nutrition in children. With the exception of the cerebellum,a tumor on one side of the brain causes symptoms and impairment on theopposite side of the body.

Compositions and methods of the present invention may be used to treat asubject who has been characterized as having cells or tissues expressingEGFRvIII, or is suspected of having cells or tissues expressingEGFRvIII. For example, subjects benefiting from treatment according tothe invention include subjects with a glioma, or subjects suspected ofhaving a glioma, for example, as evidenced by the presence of one ormore of headaches, nausea and vomiting, seizures, loss of vision, pain,weakness, numbness in the extremities, and/or cranial nerve disorders asa result of increased intracranial pressure. In particular embodiments,the glioma being treated is glioblastoma multiforme. In accordance withthis embodiment, the glioblastoma multiforme can be in the brain orspinal cord.

The present invention provides methods for inhibiting the proliferationor reducing an EGFRvIII-expressing cell population, the methodscomprising contacting a population of cells comprising anEGFRvIII-expressing cell with a CAR-expressing cell described herein,e.g., a T cell, that binds to the EGFRvIII-expressing cell. In aspecific embodiment, the present invention provides methods forinhibiting the proliferation or reducing the population of cancer cellsexpressing EGFRvIII, the methods comprising contacting theEGFRvIII-expressing cancer cell population with invention CAR-expressingcell described herein, e.g., a T cell, that binds to theEGFRvIII-expressing cell. In another embodiment, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing EGFRvIII, the methods comprisingcontacting the EGFRvIII-expressing cancer cell population with anEGFRvIII CART cell of the invention that binds to theEGFRvIII-expressing cell. In certain embodiments, the EGFRvIII CART cellof the invention reduces the quantity, number, amount or percentage ofcells and/or cancer cells by at least 25%, at least 30%, at least 40%,at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, orat least 99% in a subject with or animal model glioma or another cancerassociated with EGFRvIII-expressing cells relative to a negativecontrol. In one aspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disorder associated with EGFRvIII-expressing cells(e.g., a glioblastoma), the methods comprising administering to asubject in need an EGFRvIII CART cell described herein that binds to theEGFRvIII-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with EGFRvIII-expressing cells, the methods comprisingadministering to a subject in need thereof an EGFRvIII CART celldescribed herein that binds to the EGFRvIII-expressing cell. In anotherembodiment, the methods comprise administering to the subject in needthereof an effective amount of an EGFRvIII CART cell described hereinthat binds to the EGFRvIII-expressing cell in combination with aneffective amount of another therapy.

In one aspect, the invention pertains to a vector comprising an EGFRvIIICAR operably linked to promoter for expression in mammalian T cells. Inone aspect, the invention provides a recombinant T cell expressing theEGFRvIII CAR for use in treating EGFRvIII-expressing tumors. Therecombinant T cell expressing the anti-EGFRvIII CAR is termed anEGFRvIII CART. In one aspect, the EGFRvIII CART of the invention iscapable of contacting a tumor cell with at least one EGFRvIII CAR of theinvention expressed on its surface such that the EGFRvIII CART isactivated in response to the antigen and the CART targets the tumor celland growth of the tumor is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a EGFRvIII-expressing tumor cell, comprising contacting the tumorcell with an EGFRvIII CAR T cell described herein such that the CART isactivated in response to the antigen and targets the cancer cell,wherein the growth of the tumor is inhibited. In one aspect, theactivated CART targets and kills the cancer cell.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subject an EGFRvIIICAR T cell described herein such that the cancer is treated in thesubject. An example of a cancer that is treatable by the EGFRvIII CAR Tcell of the invention is a cancer associated with expression ofEGFRvIII. In one aspect, the cancer associated with expression ofEGFRvIII is a glioblastoma.

In one aspect, cancer associated with EGFRvIII is selected from thegroup consisting of glioblastoma multiforme (GBM), anaplasticastrocytoma, giant cell glioblastoma, gliosarcoma, anaplasticoligodendroglioma, anaplastic ependymoma, choroid plexus carcinoma,anaplastic ganglioglioma, pineoblastoma, medulloepithelioma,ependymoblastoma, medulloblastoma, supratentorial primitiveneuroectodermal tumor, and atypical teratoid/rhabdoid tumor, non-smallcell lung carcinomas, lung, breast, prostate, ovarian, colorectal andbladder carcinoma and any combination thereof.

The invention includes a type of cellular therapy where T cells aregenetically modified to express a chimeric antigen receptor (CAR) andthe CAR T cell is infused to a recipient in need thereof. The infusedcell is able to kill tumor cells in the recipient. In some embodiments,the CAR-modified T cells are able to replicate in vivo resulting inlong-term persistence that can lead to sustained tumor control. Invarious aspects, the T cells administered to the patient, or theirprogeny, persist in the patient for at least four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the T cell to the patient.

In one aspect, the CAR-modified T cells described herein may be a typeof vaccine for ex vivo immunization and/or in vivo therapy in a mammal.In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (e.g., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art therefore the present invention isnot limited to any particular method of ex vivo expansion of the cells.Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of diseases, disordersand conditions associated with expression of EGFRvIII. In certainaspects, the cells of the invention are used in the treatment ofpatients at risk for developing diseases, disorders and conditionsassociated with expression of EGFRvIII. Thus, the present inventionprovides methods for the treatment or prevention of diseases, disordersand conditions associated with expression of EGFRvIII comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations or other drug treatments, e.g., described herein.

The present invention also provides methods for inhibiting theproliferation or reducing an EGFRvIII-expressing cell population, themethods comprising contacting a population of cells comprising anEGFRvIII-expressing cell with an EGFRvIII CART cell described hereinthat binds to the EGFRvIII-expressing cell. In a specific aspect, thepresent invention provides methods for inhibiting the proliferation orreducing the population of cancer cells expressing EGFRvIII, the methodscomprising contacting the EGFRvIII-expressing cancer cell populationwith an EGFRvIII CART cell described herein that binds to theEGFRvIII-expressing cell. In one aspect, the present invention providesmethods for inhibiting the proliferation or reducing the population ofcancer cells expressing EGFRvIII, the methods comprising contacting theEGFRvIII-expressing cancer cell population with an EGFRvIII CART celldescribed herein that binds to the EGFRvIII-expressing cell. In certainaspects, the EGFRvIII CART cell of the invention reduces the quantity,number, amount or percentage of cells and/or cancer cells by at least25%, at least 30%, at least 40%, at least 50%, at least 65%, at least75%, at least 85%, at least 95%, or at least 99% in a subject with oranimal model for glioma or another cancer associated withEGFRvIII-expressing cells relative to a negative control. In one aspect,the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with EGFRvIII-expressing cells(e.g., glioblastoma), the methods comprising administering to a subjectin need an EGFRvIII CART cell described herein that binds to theEGFRvIII-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with EGFRvIII-expressing cells, the methods comprisingadministering to a subject in need thereof an EGFRvIII CART celldescribed herein that binds to the EGFRvIII-expressing cell. In oneaspect, the methods comprise administering to the subject in needthereof an effective amount of an EGFRvIII CART cell described hereinthat binds to the EGFRvIII-expressing cell in combination with aneffective amount of another therapy.

Combination Therapies

A CAR-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation.Exemplary immunotherapy approaches for malignant glioma are disclosed inJohnson et al. 2010 Curr Neurol Neurosci Rep 10:259-266. In someembodiments, a CAR-expressing cell described herein may be used in atreatment regimen in combination an agent targets extracellular matrixproteins, such as tenscin, e.g., an anti-tenascin antibody, e.g., a²¹¹At-labeled anti-tenascin antibody. In some embodiments, aCAR-expressing cell described herein may be used in a treatment regimenin combination with an immunomodulatory agent, such as interferon alpha,interferon beta, TGF-β2 peptide inhibitor, or poly-ICLC. In someembodiments, a CAR-expressing cell described herein may be used in atreatment regimen in combination with a WT1 transcription factor peptidevaccine, such as that described in Izumoto et al. 2008 J Neurosurg108:963-971.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an alkylating agent, a platinum based agent, anangiogenesis inhibitor (e.g., a VEGF pathway inhibitor, a tyrosinekinase inhibitor (e.g., an EGF pathway inhibitor), an mTOR inhibitor.

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Exemplary alkylating agents include, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®,Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracilnitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®,Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treanda®).

Exemplary platinum based agents include, without limitation,carboplatin, cisplatin, and oxaliplatin.

Exemplary angiogenesis inhibitors include, without limitation A6(Angstrom Pharmacueticals), ABT-510 (Abbott Laboratories), ABT-627(Atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (AbbottLaboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation),AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788(Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Prinomastat) (AgouronPharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP(ALN-VSP O2) (Alnylam Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen),Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573(Ridaforolimus/MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197(ArQule), ASA404 (Novartis/Antisoma), Atiprimod (CallistoPharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals),AV-951 (Aveo Pharmaceuticals), Avastin (Bevacizumab) (Genentech),AZD2171 (Cediranib/Recentin) (AstraZeneca), BAY 57-9352 (Telatinib)(Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer IngelheimPharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals),BMS-275291 (Bristol-Myers Squibb), BMS-582664 (Brivanib) (Bristol-MyersSquibb), BMS-690514 (Bristol-Myers Squibb), Calcitriol, CCI-779(Torisel) (Wyeth), CDP-791 (ImClone Systems), Ceflatonin(Homoharringtonine/HHT) (ChemGenex Therapeutics), Celebrex (Celecoxib)(Pfizer), CEP-7055 (Cephalon/Sanofi), CHIR-265 (Chiron Corporation),NGR-TNF, COL-3 (Metastat) (Collagenex Pharaceuticals), Combretastatin(Oxigene), CP-751,871(Figitumumab) (Pfizer), CP-547,632 (Pfizer),CS-7017 (Daiichi Sankyo Pharma), CT-322 (Angiocept) (Adnexus), Curcumin,Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), E7820 (EisaiLimited), E7080 (Eisai Limited), EMD 121974(Cilengitide) (EMDPharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar(Simcere), Erbitux (ImClone/Bristol-Myers Squibb), EZN-2208 (EnzonPharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme),Genistein, GSK1363089(Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib)(GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B(Ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3(ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (IntrogenTherapeutics), Iressa (ZD1839/Gefitinib), LBH589 (Faridak/Panobinostst)(Novartis), Lucentis (Ranibizumab) (Genentech/Novartis), LY317615(Enzastaurin) (Eli Lilly and Company), Macugen (Pegaptanib) (Pfizer),MEDI522 (Abegrin) (MedImmune), MLN518(Tandutinib) (Millennium),Neovastat (AE941/Benefin) (Aeterna Zentaris), Nexavar (Bayer/Onyx), NM-3(Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358(Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (PalomaPharmaceuticals, Inc.), Panzem Capsules (2ME2) (EntreMed), Panzem NCD(2ME2) (EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88(Progen Industries/Medigen Biotechnology), PKC412 (Novartis), PolyphenonE (Green Tea Extract) (Polypheno E International, Inc), PPI-2458(Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib)(Novartis), PXD101 (Belinostat) (CuraGen Corporation), RAD001(Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506)(Bayer), Revlimid (Celgene), Retaane (Alcon Research), SN38 (Liposomal)(Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230(Pasireotide)(Novartis), Squalamine (Genaera), Suramin, Sutent (Pfizer), Tarceva(Genentech), TB-403 (Thrombogenics), Tempostatin (CollardBiopharmaceuticals), Tetrathiomolybdate (Sigma-Aldrich), TG100801(TargeGen), Thalidomide (Celgene Corporation), Tinzaparin Sodium, TKI258(Novartis), TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap(Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (RegeneronPharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib(Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis),XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat(Merck), and ZSTK474.

Exemplary Vascular Endothelial Growth Factor (VEGF) receptor inhibitorsinclude, but are not limited to, Bevacizumab (Avastin®), axitinib(Inlyta®); Brivanib alaninate (BMS-582664,(S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate);Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®);Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevec®);Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanibdihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS649735-46-6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate(AMG706, CAS 857876-30-3,N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide,described in PCT Publication No. WO 02/066470); Dovitinib dilactic acid(TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3);Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS 111358-88-4);N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide(BMS38703, CAS 345627-80-7);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514);N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8);4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide(BHG712, CAS 940310-85-0); and Aflibercept (Eylea®).

Exemplary EGF pathway inhibitors include, without limitation tyrphostin46, EKB-569, erlotinib (Tarceva®), gefitinib (Iressa®), erbitux,nimotuzumab, lapatinib (Tykerb®), cetuximab (anti-EGFR mAb),¹⁸⁸Re-labeled nimotuzumab (anti-EGFR mAb), and those compounds that aregenerically and specifically disclosed in WO 97/02266, EP 0 564 409, WO99/03854, EP 0 520 722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S.Pat. No. 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983and WO 96/33980. Exemplary EGFR antibodies include, but are not limitedto, Cetuximab (Erbitux®); Panitumumab (Vectibix®); Matuzumab(EMD-72000); Trastuzumab (Herceptin®); Nimotuzumab (hR3); Zalutumumab;TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS946414-09-1). Exemplary Epidermal growth factor receptor (EGFR)inhibitors include, but not limited to, Erlotinib hydrochloride(Tarceva®), Gefitnib (Iressa®);N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514); Canertinib dihydrochloride (CI-1033);6-[4-[(4-Ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-Pyrrolo[2,3-d]pyrimidin-4-amine(AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569);Afatinib (BIBW2992); Neratinib (HKI-272);N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamicacid, (3S)-3-morpholinylmethyl ester (BMS599626);N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8); and4-[4-[[(1R)-1-Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol(PKI166, CAS 187724-61-4).

Exemplary mTor inhibitors include, without limitation, rapamycin(Rapamune®), and analogs and derivatives thereof; SDZ-RAD; Temsirolimus(Torisel®; also known as CCI-779); Ridaforolimus (formally known asdeferolimus,(1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD001);Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301-51-3);(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126, CAS 936487-67-1).

Exemplary Phosphoinositide 3-kinase (PI3K) inhibitors include, but arenot limited to,4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036082 and WO 09/055730);2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806);4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine(also known as BKM120 or NVP-BKM120, and described in PCT PublicationNo. WO2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6);(5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione(GSK1059615, CAS 958852-01-2);(1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione(PX866, CAS 502632-66-8); and 8-Phenyl-2-(morpholin-4-yl)-chromen-4-one(LY294002, CAS 154447-36-6). Exemplary Protein Kinase B (PKB) or AKTinhibitors include, but are not limited to.8-[4-(1-Aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one(MK-2206, CAS 1032349-93-1); Perifosine (KRX0401);4-Dodecyl-N-1,3,4-thiadiazol-2-yl -benzenesulfonamide (PHT-427, CAS1191951-57-1);4-[2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol(GSK690693, CAS 937174-76-0);8-(1-Hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one(palomid 529, P529, or SG-00529); Tricirbine(6-Amino-4-methyl-8-(β-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine);(αS)-α-[[[5-(3-Methyl-1H-indazol-5-yl)-3-pyridinyl]oxy]methyl]-benzeneethanamine(A674563, CAS 552325-73-2);4-[(4-Chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinamine(CCT128930, CAS 885499-61-6); 4-(4-Chlorophenyl)-4-[4-(1Hpyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and Archexin(RX-0201, CAS 663232-27-7).

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMPATH. In one aspect, the cell compositionsof the present invention are administered following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan. For example,in one embodiment, subjects may undergo standard treatment with highdose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like.Accordingly, the methods described herein can comprise administering aCAR-expressing cell described herein to a subject and furtheradministering an agent to manage elevated levels of a soluble factorresulting from treatment with a CAR-expressing cell. In one embodiment,the soluble factor elevated in the subject is one or more of IFN-γ,TNFα, IL-2 and IL-6. Therefore, an agent administered to treat this sideeffect can be an agent that neutralizes one or more of these solublefactors. Such agents include, but are not limited to a steroid, aninhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFαinhibitor is entanercept. An example of an IL-6 inhibitor is Tocilizumab(toc).

In one embodiment, the subject can be administered an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, insome embodiments, decrease the ability of a CAR-expressing cell to mountan immune effector response. Examples of inhibitory molecules includePD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 andTGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition atthe DNA, RNA or protein level, can optimize a CAR-expressing cellperformance. In embodiments, an inhibitory nucleic acid, e.g., aninhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can beused to inhibit expression of an inhibitory molecule in theCAR-expressing cell. In an embodiment the inhibitor is an shRNA. In anembodiment, the inhibitory molecule is inhibited within a CAR-expressingcell. In these embodiments, a dsRNA molecule that inhibits expression ofthe inhibitory molecule is linked to the nucleic acid that encodes acomponent, e.g., all of the components, of the CAR. In one embodiment,the inhibitor of an inhibitory signal can be, e.g., an antibody orantibody fragment that binds to an inhibitory molecule. For example, theagent can be an antibody or antibody fragment that binds to PD1, PD-L1,PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 andMDX-101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab(IgG2 monoclonal antibody available from Pfizer, formerly known asticilimumab, CP-675,206).).

PD1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD1 (Freeman et a. 2000 JExp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter etal. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitorsof PD1, PD-L1 and PD-L2 are available in the art and may be usedcombination with a CD123 CAR described herein. For example, nivolumab(also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is afully human IgG4 monoclonal antibody which specifically blocks PD-1.Nivolumab (clone 5C4) and other human monoclonal antibodies thatspecifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 andWO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD-1Pidilizumab and other humanizedanti-PD1 monoclonal antibodies are disclosed in WO2009/101611.Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG4monoclonal antibody that binds to PD1. Lambrolizumab and other humanizedanti-PD1 antibodies are disclosed in U.S. Pat. No. 8,354,509 andWO2009/114335. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1monoclonal antibody that binds to PD-L1. MDPL3280A and other humanmonoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743and U.S Publication No.: 20120039906. Other anti-PD-L1 binding agentsinclude YW243.55.S70 (heavy and light chain variable regions are shownin SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referredto as BMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD1 and B7-H1. Other anti-PD1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649. The agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., the polypeptide that isassociated with a positive signal is CD28, ICOS, and fragments thereof,e.g., an intracellular signaling domain of CD28 and/or ICOS. In oneembodiment, the fusion protein is expressed by the same cell thatexpressed the CAR. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T cell that does not express ananti-EGFRvIII CAR.

In one embodiment, the agent which enhances activity of a CAR-expressingcell described herein is miR-17-92.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T cell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988).

In certain aspects, it may be desired to administer activated T cells toa subject and then subsequently redraw blood (or have an apheresisperformed), activate T cells therefrom according to the presentinvention, and reinfuse the patient with these activated and expanded Tcells. This process can be carried out multiple times every few weeks.In certain aspects, T cells can be activated from blood draws of from 10cc to 400 cc. In certain aspects, T cells are activated from blood drawsof 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the T cell compositions of the presentinvention are administered by i.v. injection. The compositions of Tcells may be injected directly into a tumor, lymph node, or site ofinfection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates may be expanded by methods known in the art and treated suchthat one or more CAR constructs of the invention may be introduced,thereby creating a CAR T cell of the invention. Subjects in need thereofmay subsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR T cells of the present invention. In anadditional aspect, expanded cells are administered before or followingsurgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1 Redirected Autologous T Cells Engineered to ExpressEGFRvIII-Targeted Chimeric Antigen Receptor in Patients Diagnosed withEGFRvIII+ Glioblastoma

The following experiments were designed to address whether human T cellsre-directed to the surface protein EGFRvIII with an antibody-basedchimeric antigen receptor (CAR) would be effective in eliminating anEGFRvIII+ model of glioblastoma multiforme in NSG mice. In addition,experiments were designed to evaluate engraftment and persistence ofthese cells. Three different forms of CARs are tested, encompassing twodifferent single-chain variable fragments (the portion of the CARbinding to the EGFRvIII antigen), and the intracellular signalingdomains (4-1BB and CD3 zeta with and without CD28).

The immunodeficient NOD/scid/γcnull (NSG) mouse is an excellentxenotransplantation model to engraft human tumor cell lines (the braintumor line U87, which are EGFR+ and has versions engineered to beEGFRvIII+) and human T cells. Following engraftment, the human T cellscan be maintained in NSG mice for approximately 2 months, or until fatalxenogeneic GVHD (xGVHD) develops, which depends on the dose and donor ofhuman T cells infused.

Briefly, a novel CAR (3C10 CAR) using the lentiviral platform wascreated incorporating a scFv derived from anti-EGFRvIII monoclonalantibody 3C10. This CAR has been tested in vitro and in xenogeneic mousemodels. NOD/scid/γc(−/−) (NSG) mouse models have been widely used forpre-clinical assessments of CAR therapy, including evaluation oflong-term persistence of infused human T-cells. NSG mice bearing Day 7U87-EGFRvIII tumors in the brain received i.p. injections oftemozolomide (1 mg/dose) daily on Days 7-11 and i.v. infusions of: 2×10⁶human T-cells ex vivo transduced with 3C10 CAR or mock enhance greenfluorescence protein (EGFP)-vector on Days 7 and 17. On Day 21, BLIsignals were undetectable in all mice that received CAR-transducedT-cells, while mice treated with the mock-transduced T-cells showregrowth of the tumor in 4 of 5 mice following the transient anti-tumoreffect by temozolomide. In a separate experiment, mice treated withCAR-T-cells were sacrificed on Day 21, and the infiltration ofCAR-transduced T-cells was evaluated by immunohistochemistry usingbiotin-conjugated anti-F(ab′)₂ mAb (specific for the 3C10CAR) andstreptavidin-Phycoerythrin (PE). i.v. infused CAR-T-cells appeared toheavily infiltrate the tumor based on the intense PE signals while thecontrol tissue stained with streptavidin-PE but without the anti-F(ab′)₂mAb showed background signals only.

The materials and methods employed in these experiments are now.

Materials and Methods

NSG Mouse Model

A colony of immunodeficient NOD/scid/γcnull (NSG) mice was recentlyestablished. NSG mice lack T and B cells, natural killer cells, and alsohave impaired dendritic cell function. It has been confirmed thatengraftment of activated T cells was superior in NSG mice over theprevious NOD/scid/β2Mnull mouse model. Therefore the NSG model was usedfor the human xenotransplantation experiments.

Structure and Characteristics of the Biological System

Although many of the monoclonal and polyclonal Abs directed againstEGFRvIII have cross reactivity to wild type EGFR or other non-specificproteins, a monoclonal antibody (mAb) 3C10, which was originallydeveloped by immunization of mice with a 14 amino acid peptide includingthe EGFRvIII-specific fusion junction, demonstrated highly specificrecognition of EGFRvIII with negligible detectable binding to wild-typeEGFR (Okamoto et al., 1996 Br J Cancer 73:1366-1372). A research-gradelentiviral vector was used for the transduction of the T cells.

Cell Preparation for Mouse Infusion

The cells for infusion into mice are human T cells. Human mononuclearcell enriched apheresis products are obtained by leukapheresis ofhealthy volunteer donors by the University of Pennsylvania HumanImmunology Core. All specimens are collected under a UniversityInstitutional Review Board-approved protocol, and informed writtenconsent is obtained from each donor. T cells are negatively selectedusing a RosetteSep human T cell enrichment cocktail (StemcellTechnologies, Vancouver, Canada). T cells are transferred to TRPlaboratory where they are activated with research grade CD3/28 beads andexpanded in RPMI with Glutamine, 10% FBS, 20 mM Hepes, 100 U/mlPenicillin and 100 ug/ml Streptomycin. Vector transduction occurs 24hours later with packaged lentivectors added directly to activatedcultures. Cells are debeaded on day 5 and expansion is monitored with aCoulter Multisizer 3(Beckman Coulter, Fullerton, Calif.) for changes insize (fl) and total cell counts, maintaining concentration between 0.7E6to 2E6 cells/ml. Transduction efficiency for CAR-transduced T cells istested by flow cytometry by staining with either goat anti-mouseantibody (GAM, for 3C10-based CARs) or goat-anti-human (GAH, for139-based CAR). Mice are infused with 1 million CAR+ T cells per mouseby tail vein injection on day 0 of the study.

Temozolomide (TMZ) Treatment

Mice bearing i.c. U87-EGFRvIII tumor and receiving CAR+ T cells on day 0subsequently receive intraperitoneal (i.p.) injections of TMZ on days0-4 (daily for 5 days): TMZ is resolved in DMEM at 6.67 mg/ml. Eachmouse receives 50 uL TMZ solution (333 microgram/dose; approximately 17mg/kg/dose) by i.p. injections.

Clinical Grade CART

The CART-EGFRvIII T cells are prepared in the clinical cell and vaccineproduction facility (CVPF), and the cell product is autologous Tlymphocytes. CD3+ T-cells are enriched from a leukapheresis product bydepletion of monocytes via counterflow centrifugal elutriation. On day0, the manufacturing process is initiated with activation of theenriched T-cells using anti-CD3/CD28 mAb coated magnetic beads. The Tcell culture is exposed to the EGFRvIII CAR lentivirus vector andexpanded. The T-cell manufacturing process initiates in a static tissueculture (day 0 to day 5), followed by transfer to a Wave bioreactor ifneeded for additional expansion under perfusion conditions. At the endof the culture, cells are depleted of magnetic beads, washed,concentrated, and cryopreserved. The modified T cell product iscryopreserved in cryobags in a volume dependent on the cell number (at afinal concentration of maximum 10⁸/ml) using a controlled-rate freezer.Cryopreserved EGFRvIII CAR T-cell products are stored in a monitoredfreezer at ≦−130° C. The results of the experiments are now described.

Eradication of Intracranial EGFRvIII-Expressing Glioblastoma byCAR-T-Cells

Glioblastoma (GBM) is the most common and the most malignant primarybrain tumors, and responsible for approximately 12,000 cancer-relateddeaths in the US each year. Patients with GBM have a median survival ofsorter than 15 months following treatment with a combination ofchemotherapy (temozolomide) with radiotherapy (RT). Adoptive celltransfer (ACT) therapy with autologous T-cells, especially with T-cellstransduced with chimeric antigen receptors (CARs), has shown promise inrecent hematologic cancer trials. ACT with CART cells may beparticularly suitable for patients with GBM because the specificity,number, and functional phenotype of cells prepared ex vivo can bemanipulated and controlled better than native T-cells induced by in vivoimmunization.

Epidermal growth factor receptor variant III (EGFRvIII) is the mostcommon variant of the EGFR observed in human tumors but is rarelyobserved in normal tissue. This protein results from the in-framedeletion of exons 2-7 and the generation of a novel glycine residue atthe junction of exons 1 and 8 within the extra-cellular domain of theEGFR, thereby creating a tumor-specific epitope. EGFRvIII is expressedin 24% to 67% of GBM, but not in normal tissues.

To develop effective CAR therapy for GBM, three novel lentiviral CARconstructs targeting EGFRvIII were generated. Each of these vectorsencode a single-chain variable fragment (scFv) derived fromEGFRvIII-specific murine monoclonal antibodies (mAbs) 3C10 orEGFRvIII-specific humanized monoclonal antibodies (mAbs) designated“139” (FIG. 6). The 3C10 scFv was coupled with CD8a hinge, 4-1BB andCD3ζ domains with or without CD28 trans-membrane and intracellulardomains (3C10BBz28-CAR and 3C10BBz-CAR, respectively). The 139 scFv wascoupled with CD8a hinge, 4-1BB and CD3ζ domains (139BBz-CAR). HumanT-cells transduced with each of these CARs demonstrated specific andpotent lysis of EGFRvIII-expressing U87 human GBM cells (U87-EGFRvIII);see FIGS. 7A and 7B. Immunocompromised NOD/scid/γc(−/−) (NSG) micebearing Day 7 U87-EGFRvIII tumors in the brain received intravenousinfusions of 1×10⁶ human T-cells transduced ex vivo with: 1) 139BBz-CAR;2) 3C10BBz-CAR; 3) 3C10BBz28-CAR; 4) control CD19BBz-CAR targeting humanCD 19. These mice also received intraperitoneal injections oftemozolomide (330 mcg/dose) daily on Days 7-11. The tumor growth wasmonitored by bioluminescence imaging (BLI) as the U87-EGFRvIII cellsalso express luciferase. All mice treated with only saline died by Day21 due to rapid tumor growth, and temozolomide treatment without ACTinhibited but did not eradicate the U87-EGFRvIII tumors. Mice receivingCD19BBz-CAR-T-cells and temozolomide demonstrated some allogeneicresponses against U87-EGFRvIII, but the tumors continued to grow inthese mice. On the other hand, in all mice receiving 139BBz-CAR-,3C10BBz-CAR-, or 3C10BBz28-CAR-transduced T-cells, the BLI signalsdiminished to under baseline levels by Day 21, suggesting total tumoreradication (FIG. 8). Importantly, mice receiving 3C10BBz-CART cellscleared the tumor faster than either the 3C10BBz28 or 139BBz CART cells,suggesting the combination of 3C10 with BBz might afford a betterresponse in patients. The tumor growth and peripheral immune responseswere monitored to determine whether any of the three EGFRvIII-CARvectors are superior to the others for long-term anti-tumor effects.

The results presented herein strongly support development of a Phase Iclinical trial of ACT with EGFRvIII-targeting CAR-T-cells in GBMpatients who concurrently receive standard of care chemotherapy withtemozolomide.

Clinical Design

A single-arm open-label pilot study was designed to determine thesafety, tolerability and engraftment potential of CART-EGFRvIII T cellsin patients with EGFRvIII+ newly diagnosed GBMs. Generally, all subjectsare dosed with autologous CART-EGFRvIII T cells. Eligible subjects areleukapheresed to obtain large numbers of peripheral blood mononuclearcells (PBMC) for CART-EGFRvIII manufacturing. The T cells are purifiedfrom the PBMC, transduced with the humanized 3C10-CAR lentiviral vector,expanded in vitro and cryopreserved in appropriate dose aliquots. Cellsto be infused are thawed at the bedside immediately prior to infusion onday 0.

Subjects are subjected to blood tests to assess safety, and engraftmentand persistence of the CART EGFRvIII cells at regular intervals throughweek 4 (day 28). The subsets of circulating T-cells that contain the3C10-CAR vector are assessed at various times after infusion andcompared to the baseline sample. After day 28, subjects are evaluatedmonthly until 6 months with a medical history, a physical examination,brain MRI and blood tests or as per standard of care.

Research blood tests are conducted concurrent with these visits. Afterthe six months, patients are followed every 2 months for two years.After the two-year timepoint, subjects enter a roll-over study forannual follow-up by phone and questionnaire for an additional thirteenyears to assess for the diagnosis of long-term health problems, such asdevelopment of new malignancy, as required by FDA regulations pertainingto gene transfer studies.

Without wishing to be bound by any particular theory, it is believedthat because of the highly restricted expression of the EGFRvIIIprotein, there is no anticipation of any kind of off-tumor on-targetactivation of T cells. Preferably, only one infusion of theCART-EGFRvIII is administered, and therefore do not anticipateallergic-type responses either. However, one toxicity that may beencountered is bystander inflammation from T cell activation at the siteof tumor. Symptoms and signs of brain edema will be closely monitoredand managed. In some embodiments, bystander inflammation from T cellactivation can be treated by administration of an anti-inflammatoryagent, such as a steroid agent.

Example 2 Co-transduction of miR-17-92 Enhances Anti-tumor Activity ofT-Cells Transduced with the Anti-EGFRvIII Chimeric Antigen Receptor inMice Bearing Human Glioblastoma Xenografts

miR-17-92 expression confers type-1 phenotype and enhanced survival ofT-cells. It has been reported that that miR-17-92 is down-regulated inT-cells derived from glioblastoma (GBM) patients. To improve theefficacy of adoptive transfer therapy against GBM using T-cellstransduced with Chimeric Antigen Receptors (CAR-T-cells), a novellentiviral vectors for miR-17-92 and a CAR consisting of epidermalgrowth factor receptor variant III (EGFRvIII)-specific single-chainvariable fragment (scFv) coupled to the T-cell receptor CD3ζ chainsignaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 intandem (pELNS-3C10-CAR) was constructed. In addition to antigen-specificand potent cytotoxic activities against U87 GBM cells stably expressingEGFRvIII (U87-EGFRvIII), CAR-T-cells co-transduced with miR-17-92exhibited improved resistance to T-cell suppressing effects oftransforming growth factor (TGF)-β and temozolomide compared withCAR-T-cells without miR-17-92 co-transduction. In mice bearingintracranial U87-EGFRvIII xenografts, CAR-T-cells with or withouttransgene-derived miR-17-92 expression demonstrated similar levels ofpotent therapeutic effects without demonstrating any uncontrolled growthof CAR-T-cells. However, when these mice were re-challenged withU87-EGFRvIII cells in the brains, mice receiving co-transducedCAR-T-cells exhibited improved protection compared with mice treatedwith CAR-T-cells without miR-17-92 co-transduction. These data supportmiR-17-92 can be integrated in the CAR to improve the efficacy inpatients with GBM.

The results of the experiments are now described.

Construction of Lentiviral Vectors for EGFRvIII-Specific CAR andmiR-17-92

A lentiviral vector for a CAR that recognizes the EGFRvIII through asingle-chain variable fragment (scFv) derived from humanEGFRvIII-specific monoclonal antibody (mAb) 3C10 (pELNS-3C10-CAR wasgenerated (See FIG. 1A). In this construct, the EF-1α promoter drivesthe CAR fusion protein integrating the 3C10-derived scFv, CD28trans-membrane (TM) as well as 4-1BB and intracellular domains (ICD) andCD3ζ domains. A lentiviral miR-17-92 construct using the FG12-basedself-inactivating (SIN) vector (FG12-EF1a-miR-17/92 was also created(See FIG. 1B). In this vector, the EF-1αpromoter drives miR-17-92 andthe human UbiC promoter drives enhanced green fluorescence protein(EGFP) marker gene for tracking of transduced cells. Abbreviations usedin the schema: RSV/HIV-1 5′LTR; Hybrid RSV promoter-R/U5 long terminalrepeat, EF-1α; Human elongation factor 1α-subunit promoter, VH; Variableregion in the heavy chain of the 3C10 immunoglobulin, VL; Variableregion in the light chain of the 3C10 immunoglobulin, HIV-1 Δ-3′LTR;Self-inactivating 3′ long terminal repeat with deletion in U3 region,CMV/HIV-1 5′LTR Hybrid CMV promoter-R/U5 long terminal repeat, UbiC;Ubiquitin C promoter.

In Vitro Characterization of Human T-Cells Transduced with the CAR andmiR-17-92

Healthy donor-derived CD3⁺ T-cells were transduced with pELNS-3C10-CAR,and the cells were evaluated for expression levels of the transgene byflow cytometry for expression of 3C10-CAR and miR-17-92 by anti-mouse(Fab′)₂ antibody and EGFP, respectively (FIG. 2A, Left). Usinganti-mouse F(ab′)₂ Ab, which is specific for the 3C10-derived scFv onhuman T-cells, nearly half (48.9%) of the T-cells expressing the3C10-derived scFv on their surface were detected.

To obtain human T-cells expressing both the CAR and transgene-derivedmiR-17-92, CD3⁺ T-cells were co-transduced with pELNS-3C10-CAR andFG12-EF1a-miR-17/92 by sequential infection of the two lentiviralvectors. At 24 hours after the initial transduction with pELNS-3C10-CAR,the T-cells were transduced with FG12-EF1a-miR-17-92. It was observedthat approximately a quarter (23.6%) of the total T-cells expressed bothCAR and EGFP (FIG. 2A, Right). For subsequent in vitro studies,CAR-transduced T-cells (CAR-T-cells) were enriched using biotinylatedanti-mouse F(ab′)₂ Ab and anti-biotin MACS. Based on the efficiency ofco-transduction (FIG. 2A, Right), at least 50% of the CAR-T-cells alsoexpressed EGFR (hence the transgene-derived miR-17-92). By real-timePCR, 3-4 fold higher expression of miR-17-92 was detected in the F(ab′)₂Ab-enriched, miR-17-92-co-transduced CAR-T-cells compared with T-cellstransduced with the CAR alone (FIG. 2B). FIG. 2B demonstrates theexpression levels of the miR-17-92 cluster members, miR-17-3p, miR-17-5pand miR-92a-1 in transduced T cells measured by qRT-PCR. Mean±SD valuesof 3 replicate measurements from one of three experiments with similarresults are depicted. * indicates p<0.05 between the two groups usingstudent t test. FIG. 2C depicts EGFRvIII specific cytotoxic activitiesof transduced T cells evaluated by a 12-h 51Cr-release assay at variousE:T ratios against 51Cr-labeled U87-EGFRvIII or control U87 cells.Control cells were Mock (EGFP)-transduced T-cells. Values indicatemean±SD in triplicated wells.

While the mock-transduced T-cells showed only background levels of lysisagainst both parental U87 (EGFRvIII-negative) and U87-EGFRvIII cells,T-cells transduced with the CAR demonstrated potent and specific lysisof EGFRvIII-expressing U87 human GBM cells (U87-EGFRvIII) with onlybackground levels of cytotoxic effects against parental U87 cells (FIG.2C). In these 12h ⁵¹ Cr-release assays, co-transduction of CAR-T-cellswith miR-17-92 did not significantly enhance their specific cytotoxicactivity against U87-EGFRvIII target cells.

miR-17-92 Co-transduction Confers Enhanced IFN-γ release and Resistanceto Suppressive Effects by TGF-β and Temozolomide (TMZ—Standard of CareTherapy)

In a previous study (Sasaki et al., 2010, J. Transl Med 8:17), CD4⁺ Tcells derived from miR-17-92 transgenic mice demonstrated increasedIFN-γ production when compared with counterparts derived from wild typemice; and transfection of human Jurkat T cells with miR-17-92 lead toenhanced resistance to activation-induced cell death (AICD).

Experiments were conducted to evaluate whether co-transduction ofCAR-T-cells with miR-17-92 confers improved IFN-γ production, cellproliferation and lesser degrees of apoptotic death when they areexposed to a chemotherapy agent TMZ or an immuno-suppressive cytokineTGF-β.

When CAR-T-cells were stimulated with EGFRvIII-transduced artificialAntigen-Presenting Cells (aAPCs) without TGF-β or TMZ, the cellsexpressed similar levels of IFN-γ with or without co-transduction.However, when the cells were exposed to escalating doses of TGF-β orTMZ, CAR-T-cells without miR-17-92 co-transduction producedsignificantly reduced levels of IFN-γ, while the co-transducedCAR-T-cells maintained high level production of IFN-γ (FIG. 3A). Openbars and closed bars represent results from CAR-T-cells (withoutmiR-17-72) and miR-17-92 co-transduced CAR-T cells, respectively. FIG.3A shows IFN-γ produced by the transduced T cells during the 25 last 24h of 96 h co-culture. FIG. 3B shows relative proliferation levelsbetween the groups were evaluated by WST1 assay following the 3-dayco-culture course. FIGS. 3C and 3D show apoptotic death of CAR-T-cellsevaluated by Annexin-V and PI. FIG. 3C show mean fluorescent intensityfor Annexin-V on CAR-T-cells exposed to TMZ. Values indicate mean±SD intriplicate wells. (* indicates P<0.05) FIG. 3D show flow cytometrichistograms for Annexin-V+ and/or PI+ in one of the three experimentswith similar results.

Experiments were conducted to evaluate the effects of miR-17-92co-transduction on proliferation of CAR-T-cells in the presence of TMZin culture. Experiments were designed to induce the proliferation ofCAR-T-cells with EGFRvIII-expressing aAPC and the proliferation wasevaluated by WST-1 assay (FIG. 3B). Without TMZ, miR-17-92-co-transducedCAR-T-cells demonstrated a trend toward a faster proliferation ratecompared with control CAR-T-cells, but the difference was notsignificant. To specifically evaluate the impact of TMZ on theCAR-T-cell proliferation, in FIG. 3B, the proliferation rate of thecells in each group was depicted relative to the proliferation of thesame cells without TMZ. When increasing concentrations of TMZ are addedin the culture, the degrees of growth suppression was significantly lessin the miR-17-92 co-transduced CAR-T-cells compared with the controlCAR-T-cells.

Experiments were conducted to evaluate whether miR-17-92-co-transductionwould render CAR-T-cells more resistant to TMZ-induced apoptosis. Tothis end, flow-cytometric assessments of Annexin V⁺ and propidium iodide(PI)⁺ CAR-T-cells were conducted in increasing concentrations of TMZ(FIGS. 3C and 3D). It was observed that a dose-dependent increase ofboth early apoptotic (Annexin V⁺PI⁻), apoptotic/necrotic (Annexin V⁺PI⁺)and necrotic (Annexin V⁻PI⁺) cells, and miR-17-92-co-transducedCAR-T-cells demonstrated lesser degrees of the apoptotic changescompared with control CAR-T-cells.

Intravenous Injection of CAR-T-Cells in Combination with TMZ Leads toComplete Remission of Established U87-EGFRvIII Tumors in NSG Mice

Experiments were conducted to evaluate the efficacy of CAR-T-cells inimmunocompromised NOD/scid/γc(−/−) (NSG) mice bearing established (Day7) intracranial U87-EGFRvIII tumors. Mice received a single intravenous(i.v.) infusion of miR-17-92 co-transduced CAR-T-cells, CAR-T-cellswithout co-transduction of miR-17-92, or mock-transduced T-cells(2×10⁶/mouse) via the tail vein. As newly diagnosed GBM patientsroutinely receive TMZ therapy, experiments were designed to administerintraperitoneal (i.p.) daily injections of TMZ for 5 days starting onthe day of T-cell infusion (FIG. 4A). FIG. 4B shows a Kaplan-Meieranalysis. Median survival of the mice treated with CAR-T cells (with orwithout co-transduction of miR-17-92) was significantly greater comparedthe mice with mock transduced T cells (p<0.05). TMZ treatment itself wasineffective as all the control mice receiving TMZ and mock-transducedT-cells died within 3 weeks (day 21) after the T-cell infusion (FIG.4B). Although one of five mice with CAR-T-cells and two of five micewith miR-17-92 co-transduced CAR-T-cells died for the tumor progressionby day 22, all the other mice in these groups survived longer than 40days. Results are from one of two independent experiments with similarresults. There was not a statistically significant difference insurvival of the mice receiving miR-17-92-co-transduced CAR-T-cells vs.CAR-T-cells without miR-17-92 co-transduction (log-rank test: p=0.5485).

miR-17-92 Co-transduced CAR-T-Cells Confers a Persistent ProtectionAgainst U87-EGFRvIII Tumors in Mice

To determine whether CAR-T-cells infused in the mice in the experimentpresented in FIG. 4 can provide long-term protection of the hostsagainst the U87-EGFRvIII tumors, the survivors were re-challenged withinoculation of U87-EGFRvIII cells in the contra-lateral hemisphere ofthe brain on Day 49 (FIG. 5). While the re-challenged tumor cells grewin all three mice treated with CAR-T-cells, none of the mice treatedwith miR-17-92-co-transduced CAR-T-cells demonstrated BLI signals beyondthe background levels. These results strongly suggest thatco-transduction of miR-17-92 cluster confers long-term persistence ofthe CAR-T-cells, thereby providing prolonged protection of the hostagainst the tumor growth. Longitudinal measurements of tumor-derivedmean photon flux±SD from the 2 groups of mice. The backgroundluminescence level (up to 10^3 p/s) was defined based on the levelsobserved in non-tumor-bearing mice imaged in parallel with tumor-bearingmice in treatment groups

miR-17-92 can be Integrated in the CAR to Improve Efficacy

The results presented herein demonstrate the effects of miR-17-92co-expression in T-cells transduced with the novel anti-EGFRvIII-CAR(3C10-CAR) integrating 3C10 scFv with CD3ζ chain, CD137 (4-1BB) andCD28. The present results show that co-expression of miR-17-92 confersimproved resistance to T-cell growth-suppressing effects of TGF-β andtemozolomide. In vivo, T-cells co-transduced with both 3C10-CAR andmiR-17-92 demonstrated more persistent therapeutic effects compared withT-cells transduced with 3C10-CAR alone.

Lentiviral transduction of miR-17-92 in the present study confersectopic over-expression of the miR-cluster in transduced T-cells. Inphysiological conditions, however, expression levels of endogenousmiR-17-92 in T-cells appear to be tightly regulated. In human CD8⁺ Tcells, miR-17-92 expression is detected high levels in naïve cells butdiminishes as the cells differentiate (Salaun et al., 2011, J Transl Med9:44). In a mouse model of lymphocytic choriomeningitis virus infection,miR-17-92 is strongly up-regulated following T-cell activation, howeverdown-regulated after clonal expansion, and further silenced duringmemory development (Wu et al., 2012, Proc Natl Acad Sci USA109:9965-9970). In this referenced study, miR-17-92 is necessary for therapid T-cell expansion and their IFN-γ expression. However,overexpression of miR-17-92 skews the differentiation toward short-livedterminal effector cells. Failure to down-regulate miR-17-92 leads to agradual loss of memory cells and defective central memory celldevelopment (Wu et al., 2012, Proc Natl Acad Sci USA 109:9965-9970).These observations are not necessarily consistent with the resultspresented herein as persistence of miR-17-92-co-transduced CAR-T-cellsand their efficient ability to protect the hosts from the re-challengedU87-EGFRvIII cells was observed. Without wishing to be bound by anyparticular theory, it is believed that this observation is attributableto the combinatory effects of the co-stimulatory molecules provided inthe CAR and the miR-17-92.

Although miR-17-92 has been described as an oncogenic miR (van Haaftenand Agami, 2010, Genes & Development 24:1-4), miR-17-92 overexpressionitself is known not to be oncogenic in lymphocytes (Xiao et al., 2008,Nat Immunol 9:405-414). Indeed, uncontrolled proliferation ofmiR-17-92-transduced T-cells in the current study was not observed.Nonetheless, as an alternative approach for better safety assurance,transient transduction of T-cells with miR-17-92 itself, instead oflentiviral stable transfer, and multiple injection of those T-cells mayrepresent a reasonable approach without the associated safety concernsof integrating viral vectors (Zhao et al., 2010, Cancer Research70:9053-9061).

In regard to the EGFRvIII-targeting CARs for therapy of GBM, recently,Morgan et al. evaluated scFv sequences derived from seven differentanti-EGFRvIII mAbs, including 3C10 and human 139, in γ-retroviral CARs(Morgan et al., 2012, Hum Gene Ther 23:1043-1053). The in vitrocharacterization of those CARs revealed the 3C10 and the 139 as two ofthe three clones that yielded specific IFN-γ production in response toEGFRvIII-expressing target cells, but not cells expressing the wild-typeEGFR gene.

It is also important to recognize that EGFRvIII is expressed only in apopulation of GBM patients and fractions of the GBM cells even in“EGFRvIII-positive” cases (Heimberger et al., 2005, Clin. Cancer Res.11:1462-1466). Immunotherapy targeting EGFRvIII as the single targetwill likely result in the outgrowth of GBM cells that havedown-regulated the immunotherapy-targeted antigen (Sampson et al, 2010,J Clin Oncol 28:4722-4729). A number of previous studies have developedCARs against GBM-associated antigens, such as IL-13Rα2 (Kong et al.,2012, Clin Cancer Res 18:5949-5960; Kahlon et al., 2004, Cancer Res.64:9160-9166), HER-2 (Ahmed et al., 2010, Clinical Cancer Research16:474-485) and EphA2 (Chow, K. K. et al. T Cells Redirected to EphA2for the Immunotherapy of Glioblastoma. Mol Ther (2012). Without wishingto be bound by any particular theory, it is believed that effective CARtherapy should ultimately employ T-cells that are able to resistGBM-induced suppression mechanisms and target multiple antigens, so thatthe infused T-cells will exhibit effective and sustained therapeuticeffects against GBM with heterogenous antigen-expression profiles.

The results presented herein demonstrate the benefits of using T-cellsco-transduced with pELNS-3C10-CAR and FG12-EF1a-miR-17/92. As analternative approach to achieve co-expression of the CAR and miR-17-92transgene, a pELNS-based lentiviral vector that expresses both 3C10-CARgene and miR-17-92 gene as a single transcript was constructed. Use ofthis single “tandem” vector may have an advantage in terms of relativelysimple transduction procedures and straightforward regulatory processescompared with the two vector-based approach. Furthermore, all T-cellsthat express the CAR should also express miR-17-92. However, it wasfound that the transduction efficiency of the “tandem” vector is lowerthan that by the two vector approach likely because the titer oflentivirus decreases as the size of insert increases. As discussedelsewhere herein, lentiviral transduction of 3C10-CAR gene andelectroporation of miR-17-92 in combination may be a feasible strategy.

In the present study, it was also found that 40% to 60% of CD3⁺CAR-T-cells were CD4⁺, and that CD4⁺ CAR-T-cells effectively lysedU87-EGFRvIII cells in an EGFRvIII-specific manner. It has been reportedthat Perforin⁺ CD4⁺ T-cells mediate cytotoxic activities via theperforin/granzyme B pathway, but not the Fas/FasL pathway (Porakishviliet al., 2004, Haematologica 89:435-443). Hence, it is believed that theCD4⁺ CAR-T-cells in the current study expressed perforin and granzyme Bto mediate the observed lytic activities against U87-EGFRvIII cells.

In summary, the current study provides a strong foundation forevaluation of CAR therapy integrating miR-17-92.

Example 3 CAR Sequences

Murine monoclonal antibody (mAb) 3C10 was originally developed byimmunization of mice with a 14 amino acid peptide (PEP3) including theEGFRvIII-specific fusion junction and demonstrated highly specificrecognition of EGFRvIII without any detectable binding to wild-type EGFR(Okamoto et al, British J. Cancer 1996, 73:1366-1372). Subsequently, asingle-chain variable fragment (scFv) of mAb 3C10 was produced and cDNAfor the 3C10 scFv was obtained. While avidity and/or antigen-specificityof the original mAbs can be often lost in scFv forms, the 3C10 scFvretained its selective reactivity with the EGFRvIII-specific epitope(Nakayashiki et al., Jpn. J. Cancer Res. 2000, 91:1035-1043).

An EGFRvIII CAR was constructed by cloning the 3C10scFv (mouse) withCD28, 4-1BB, and CD3 zeta into the pELNS lentiviral backbone plasmid(EF1 promoter). Another EGFRvIII CAR was generated by cloning the3C10scFv into a CD8ahinge/CD8TM/4-1BB/CD3zeta pELNS lentiviral backbone,which is expressed by EF1a promoter.

3C10scFv-CD28BBzeta CAR (Amino Acid) (SEQ ID NO: 1)MALPVTALLLPLALLLHAARPGSEIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLEWIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAVYYCAFRGGVYWGPGTTLTVSSGGGGSGGGGSGGGGSHMDVVMTQSPLTLSVAIGQSASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGIYYCWQGTHFPGTFGGGTKLEIKASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 3C10scFv-BBz CAR (Amino Acid)(SEQ ID NO: 2) MALPVTALLLPLALLLHAARPGSEIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLEWIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAVYYCAFRGGVYWGPGTTLTVSSGGGGSGGGGSGGGGSHMDVVMTQSPLTLSVAIGQSASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLISLVS KLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGIYYCWQGTHFPGTFGGGTKLEIKASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR3C10scFv-CD28BBzeta CAR (Nucleic Acid) (SEQ ID NO: 18)atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatccgagattcagctgcagcaatctggggcagaacttgtgaagccaggggcctcagtcaagctgtcctgcacaggttctggcttcaacattgaagactactatattcactgggtgaagcagaggactgaacagggcctggaatggattggaaggattgatcctgagaatgatgaaactaaatatggcccaatattccagggcagggccactataacagcagacacatcctccaacacagtctacctgcaactcagcagcctgacatctgaggacactgccgtctattactgtgcctttcgcggtggagtctactgggggccaggaaccactctcacagtctcctcaggaggtggtggttccggtggtggtggttccggaggtggtggttcacatatggatgttgtgatgacccagtctccactcactctatcggttgccattggacaatcagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctctctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgagaatcagcagagtggaggctgaggatttgggaatttattattgctggcaaggtacacattttcctgggacgttcggtggagggaccaagctggagataaaagctagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 3C10scFv-BBz CAR (Nucleic Acid) (SEQ ID NO: 19)Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatccgagattcagctgcagcaatctggggcagaacttgtgaagccaggggcctcagtcaagctgtcctgcacaggttctggcttcaacattgaagactactatattcactgggtgaagcagaggactgaacagggcctggaatggattggaaggattgatcctgagaatgatgaaactaaatatggcccaatattccagggcagggccactataacagcagacacatcctccaacacagtctacctgcaactcagcagcctgacatctgaggacactgccgtctattactgtgcctttcgcggtggagtctactgggggccaggaaccactctcacagtctcctcaggaggtggtggttccggtggtggtggttccggaggtggtggttcacatatggatgttgtgatgacccagtctccactcactctatcggttgccattggacaatcagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctctctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgagaatcagcagagtggaggctgaggatttgggaatttattattgctggcaaggtacacattttcctgggacgttcggtggagggaccaagctggagataaaagctagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

The scFv fragment termed “139” is a human antibody to EGFRvIII (Morganet al., 2012 Hum Gene Ther 23(10): 1043-53). An EGFRvIII CAR comprisingthe 139 scFv was generated by initially synthesizing the 139 scFv. Thesequence for the 139 scFv was cloned with a leader sequence, CD8 hinge,transmembrane (TM) domain, and the desired signaling domains. Forexample, the sequence for the 139 scFv was cloned with the signalingdomains for 4-1BB and CD3 zeta. The CAR construct (139scFv-BBZ) isexpressed from the pELNS vector for lentivirus production.

139scFv-BBz CAR (Amino Acid) (SEQ ID NO: 3)MALPVTALLLPLALLLHAARPGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNNLAWYQQKPGKAPKRLIYAASNLQSGVPSRFTGSGSGTEFTLIVSSLQPEDFATYYCLQHHSYPLTSGGGTKVEIKRTGSTSGSGKPGSGEGSEVQVLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSSGWSEYWGQGTLVTVSSASTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR139scFv-BBz CAR (Nucleic Acid) (SEQ ID NO: 20)AtggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatccgacatccagatgacccagagccctagcagcctgagcgccagcgtgggcgacagagtgaccatcacctgtcgggccagccagggcatcagaaacaacctggcctggtatcagcagaagcccggcaaggcccccaagagactgatctacgctgccagcaatctgcagagcggcgtgcccagcagattcaccggaagcggctccggcaccgagttcaccctgatcgtgtccagcctgcagcccgaggacttcgccacctactactgcctgcagcaccacagctaccctctgaccagcggcggaggcaccaaggtggagatcaagcggaccggcagcaccagcggcagcggcaagcctggcagcggcgagggaagcgaggtccaggtgctggaatctggcggcggactggtgcagcctggcggcagcctgagactgagctgtgccgccagcggcttcaccttcagcagctacgccatgtcttgggtccggcaggctcctggaaagggcctggaatgggtgtccgccatcagcggctctggcggctccaccaactacgccgacagcgtgaagggccggttcaccatcagccgggacaacagcaagaacaccctgtatctgcagatgaacagcctgagagccgaggacaccgccgtgtactactgtgccggcagcagcgggtggagcgagtactggggccagggcacactggtcacagtgtctagcgctagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctCar Components

NUCLEIC ACID SEQUENCES: 3C10 scFv Nucleotide Sequence (Mouse);(SEQ ID NO: 4) GAGATTCAGCTGCAGCAATCTGGGGCAGAACTTGTGAAGCCAGGGGCCTCAGTCAAGCTGTCCTGCACAGGTTCTGGCTTCAACATTGAAGACTACTATATTCACTGGGTGAAGCAGAGGACTGAACAGGGCCTGGAATGGATTGGAAGGATTGATCCTGAGAATGATGAAACTAAATATGGCCCAATATTCCAGGGCAGGGCCACTATAACAGCAGACACATCCTCCAACACAGTCTACCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCCTTTCGCGGTGGAGTCTACTGGGGGCCAGGAACCACTCTCACAGTCTCCTCAGGAGGTGGTGGTTCCGGTGGTGGTGGTTCCGGAGGTGGTGGTTCACATATGGATGTTGTGATGACCCAGTCTCCACTCACTCTATCGGTTGCCATTGGACAATCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTCTCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTGCTGGCAAGGTACACATTTTCCTGGGACGTTCGGTGGAGGGACCAAGCTGGAGAT AAAA139 scFv Nucleotide Sequence (Humanized); (SEQ ID NO: 5)GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGGGCATCAGAAACAACCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGAGACTGATCTACGCTGCCAGCAATCTGCAGAGCGGCGTGCCCAGCAGATTCACCGGAAGCGGCTCCGGCACCGAGTTCACCCTGATCGTGTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGCACCACAGCTACCCTCTGACCAGCGGCGGAGGCACCAAGGTGGAGATCAAGCGGACCGGCAGCACCAGCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGAAGCGAGGTCCAGGTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGTCTTGGGTCCGGCAGGCTCCTGGAAAGGGCCTGGAATGGGTGTCCGCCATCAGCGGCTCTGGCGGCTCCACCAACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCGGCAGCAGCGGGTGGAGCGAGTACTGGGGCCAGGGCACACTGGTCACAGTGTCTA GCleader (nucleic acid sequence); (SEQ ID NO: 6)ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC CGCCAGGCCGhinge (nucleic acid sequence); (SEQ ID NO: 7)ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT transmembrane (nucleic acid sequence);(SEQ ID NO: 8) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB Intracellular domain (nucleic acid sequence);(SEQ ID NO: 9) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3 zeta (nucleic acid sequence); (SEQ ID NO: 10)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCD3 zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3);(SEQ ID NO: 100) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC AMINO ACID SEQUENCES:3C10 scFv Amino Sequence (Mouse); (SEQ ID NO: 11)EIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLEWIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAVYYCAFRGGVYWGPGTTLTVSSGGGGSGGGGSGGGGSHMDVVMTQSPLTLSVAIGQSASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGIYYCWQGTHFPGTFGGGTKLEIK 139 scFv Amino Sequence (Human); (SEQ ID NO: 12)DIQMTQSPSSLSASVGDRVTITCRASQGIRNNLAWYQQKPGKAPKRLIYAASNLQSGVPSRFTGSGSGTEFTLIVSSLQPEDFATYYCLQHHSYPLTSGGGTKVEIKRTGSTSGSGKPGSGEGSEVQVLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGSSGWSEYWGQGTLVTVSS leader (amino acid sequence) (SEQ ID NO: 13)MALPVTALLLPLALLLHAARP hinge (amino acid sequence) (SEQ ID NO: 14)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDtransmembrane (amino acid sequence) (SEQ ID NO: 15)IYIWAPLAGTCGVLLLSLVITLYC4-1BB Intracellular domain (amino acid sequence) (SEQ ID NO: 16)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCD3 zeta domain (amino acid sequence) (SEQ ID NO: 17)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRCD3 zeta domain (amino acid sequence; NCBI Reference Sequence NM_000734.3)(SEQ ID NO: 99) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The nucleotide encoding the polypeptide of SEQ ID NO:11 is provided asSEQ ID NO:4. The nucleotide encoding the polypeptide of SEQ ID NO:12 isprovided as SEQ ID NO:5. The nucleotide encoding the polypeptide of SEQID NO:13 is provided as SEQ ID NO:6. The nucleotide encoding thepolypeptide of SEQ ID NO:14 is provided as SEQ ID NO:7. The nucleotideencoding the polypeptide of SEQ ID NO:15 is provided as SEQ ID NO:8. Thenucleotide encoding the polypeptide of SEQ ID NO:16 is provided as SEQID NO:9. The nucleotide encoding the polypeptide of SEQ ID NO:17 isprovided as SEQ ID NO:10. The nucleotide encoding the polypeptide of SEQID NO:1 is provided as SEQ ID NO:18. The nucleotide encoding thepolypeptide of SEQ ID NO:2 is provided as SEQ ID NO:19. The nucleotideencoding the polypeptide of SEQ ID NO:3 is provided as SEQ ID NO:20. Thenucleotide encoding the polypeptide of SEQ ID NO:99 is provided as SEQID NO:100.

Example 4 Predicted CDR Designations for the EGFRvIIICAR

The predicted CDR designations for the EGFRvIII CAR under Kabat are asfollows:

VH: (SEQ ID NO: 21) EIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLEWIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAVYYCAF RGGVYWGPGTTLTVSS;;wherein CDR1 is DYYIH (SEQ ID NO: 22), CDR2 is RIDPENDETKYGPIFQG (SEQ ID NO: 23), and CDR3 is RGGVY (SEQ ID NO: 24). VL:(SEQ ID NO: 25) DVVMTQSPLTLSVAIGQSASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGIYYCWQGTHFP GTFGGGTKLEIK;;wherein CDR1 is KSSQSLLDSDGKTYLN (SEQ ID NO: 26),CDR2 is LVSKLDS (SEQ ID NO: 27), and CDR3 is WQGTH FPGT (SEQ ID NO: 28).

The predicted CDR designations for the EGFRvIII CAR under Chothia are asfollows:

VH: (SEQ ID NO: 29) EIQLQQSGAELVKPGASVKLSCTGSGFNIEDYYIHWVKQRTEQGLEWIGRIDPENDETKYGPIFQGRATITADTSSNTVYLQLSSLTSEDTAVYYCAF RGGVYWGPGTTLTVSS;;wherein CDR1 is GFNIEDY (SEQ ID NO: 30), CDR2 isDPENDE (SEQ ID NO: 31), and CDR3 is RGGVY (SEQ ID NO: 32). VL:(SEQ ID NO: 33) DVVMTQSPLTLSVAIGQSASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGIYYCWQGTHFP GTFGGGTKLEIK;;wherein CDR1 is SQSLLDSDGKTY (SEQ ID NO: 34), CDR2is LVS (SEQ ID NO: 35), and CDR3 is GTHFPG (SEQ ID NO: 36).

Example 5 Humanization of Murine Anti-EGFRvIII Antibody

Humanization of murine EGFRvIII antibody is desired for the clinicalsetting, where the mouse-specific residues may induce a human-anti-mouseantigen (HAMA) response in patients who receive treatment with T cellstransduced with the murine CAR construct. VH and VL sequences ofhybridoma derived murine EGFRvIII antibody were extracted from publishedliterature (Morgan et al. (2012) Human Gene Therapy, 23: 1043-1953,Supra). Humanization was accomplished by grafting CDR regions frommurine EGFRvIII antibody onto human germline acceptor frameworks VH1_1-for VH5_5a as well as VK2_A17 or VK4_B3 (vBASE database). In addition tothe CDR regions, several framework residues, i.e. VK2 #36, #49, VK4 #2,#36, #46, #49, VH1 #2, #24, #76, #94 and VH5 #2, #24, #73, #76, #94,thought to support the structural integrity of the CDR regions wereretained from the murine sequence. Further, the human J elements JH6 andJK4 were used for the heavy and light chain, respectively. The resultingamino acid sequences of the humanized antibody were designatedVK2_A17/Hz1 and VK4_B3/Hz1 for the light-chains and VH1_1-f/Hz1,VH5_5-a/Hz1 for the heavy chains shown in FIG. 9. The residue numberingfollows Kabat (Kabat E. A. et al, 1991, supra). For CDR definitions,both Kabat as well as Chothia et al, 1987 supra) were used. Frame workresidues retained from mouse EGFRvIII are shown boxed bold/italic, CDRresidues are underlined.

Based on the humanized light and heavy chain sequences as shown in FIG.9, a total of 8 framework combinations were used to generate solublescFv's for further validation. The order in which the VL and VH domainsappear in the scFv was varied (i.e., VL-VH, or VH-VL orientation), andfour copies of the “G₄S” subunit (SEQ ID NO: 37), in which each subunitcomprises the sequence GGGGS (SEQ ID NO:37) was used to connect theframeworks. FIG. 9 discloses the CDR's in the VH and VL sequencescalculated by Kabat et al and Chothia et al. (Supra).

Cloning:

DNA sequences coding for mouse and humanized VL and VH domains wereobtained, and the codons for the constructs were optimized forexpression in cells from Homo sapiens.

Sequences coding for VL and VH domain were subcloned into expressionvectors suitable for secretion in mammalian cells. Elements of theexpression vector include a promoter (Cytomegalovirus (CMV)enhancer-promoter), a signal sequence to facilitate secretion, apolyadenylation signal and transcription terminator (Bovine GrowthHormone (BGH) gene), an element allowing episomal replication andreplication in prokaryotes (e.g. SV40 origin and ColE1 or others knownin the art) and elements to allow selection (ampicillin resistance geneand zeocin marker).

Example 6 Characterization of Humanized Anti-EGFRvIII Soluble scFvFragments

Soluble scFv fragments were generated described above using standardmolecule biology techniques. These soluble scFvs were used incharacterization studies to examine the stability, cell surfaceexpression, and binding properties of the scFvs.

scFv Expression and Purification

For transfection of each scFv construct, approximately 3e8 293F cellswere transfected with 100 μg of plasmid using PEI as the transfectionreagent at the ratio of 3:1 (PEI:DNA). The cells were grown in 100 mlEXPi293 Expression media (Invitrogen) in a shaker flask at 37° C., 125rpm, 8% CO₂. The culture was harvested after six days and used forprotein purification.

293F cells were harvested by spinning down at 3500 g for 20 minutes. Thesupernatant was collected and filtered through VacuCap90 PF Filter Unit(w/0.8/0.2 μm Super Membrane, PALL). Around 400 ul of Ni-NTA agarosebeads (Qiagen) were added to the supernatant. The mixture was rotatedand incubated for 4 hrs at 4° C. It was loaded onto a purificationcolumn and washed with washing buffer with 20 mM Histidine. The proteinwas eluted with 500 μl elution buffer with 300 mM Histidine. The sampleswere dialyzed against PBS buffer at 4 C overnight. Protein samples werequantified using nanodrop 2000 c.

EC₅₀ by FACS Binding of Purified scFv's to Cells Expressing Either HumanEGFR Wild Type or EGFRvIII

The following experiments were conducted to demonstrate that all thehumanized EGFRvIII scFv variants have comparable binding to EGFRvIII,but no binding to wild type EGFR.

HEK293F suspension cells were transiently transfected with either wildtype hEGFR or hEGFRvIII and were harvested 2 days after transfection.Approximately 5e5 cells/per well were transferred to a BD Falcon 96 wellplate. The cells were spun down at 900 rpm (Sorval Legend XT centrifuge)for 3 minutes. The supernatant was removed. Anti-EGFRvIII scFv proteinsamples were diluted in DPBS with 5% FBS. The samples were added intothe wells, mixed and incubated for 1 hour. The cells were washed twicein the DPBS with 5% FBS. The cells were incubated with anti-poly His PE(R&D) for 1 hour, washed twice before FACS analysis (LSRII from BDBiosciences).

The EC₅₀ of mouse scFv (m3C10) for hEGFRvIII was determined to be ˜5 nMas shown in FIG. 10. All the humanized EGFRvIII scFv variants showedEC₅₀ values in the single digit to low double digit nM EC₅₀s range (5-50nM), Moreover, no appreciable binding of constructs 2173 and 2174 towild type EGFR expressing cell lines was detected indicating an improvedsafety profile compared to murine 3C10, as shown in FIG. 11. Based onthese studies, clone 2173 was selected for further clinicalcharacterization, as shown in Example 8

Example 7 Humanized EGFRvIII CAR Constructs

ScFv to be used in the final CAR constructs were derived from thehumanized framework sequences described in Example 1. The order in whichthe VL and VH domains appear in the scFv was varied (i.e., VL-VH, orVH-VL orientation). A (G4S)₄ (SEQ ID NO: 113), linker was used toconnect the variable domains to create the scFvs shown in Table 1.

TABLE 1 Humanized EGFRvIII scFv constructs showing VH and VL orientationand linker length (Table discloses “G4S” as SEQ ID NO: 37) construct IDLength aa annotation 108358 277 VH1-VK4, 4G4S 108359 277 VK4-VH1, 4G4S108360 277 VH5-VK2, 4G4S 108361 277 VK2-VH5, 4G4S 107276 277 VH1-VK2,4G4S 111046 278 VH5-VK4, 4G4S 111048 278 VK4-VH5, 4G4S 107277 277VK2-VH1, 4G4S 107275 mEGFRvIII 3C10 274 VH-VL, 3G4S + HM EGFRvIII 139269 VL-VH,

The sequences of the humanized scFv fragments are provided below inTable 2 (SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ IDNO:62, SEQ ID NO:68, SEQ ID NO:74, and SEQ ID NO:80). These scFvfragments were used with additional sequences, SEQ ID NOs: 13-17, togenerate full CAR constructs with SEQ ID NOs: SEQ ID NO:43, SEQ IDNO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ IDNO:79, and SEQ ID NO:85.

These clones all contained a Q/K residue change in the signal domain ofthe co-stimulatory domain derived from CD3zeta chain.

TABLE 2 Humanized EGFRvIII CAR Constructs SEQ  ID Name NO: SequenceCAR 1 CAR1 38 eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetscFv kygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssggggsgdomain gggsggggsggggsdvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkv eik CAR139 gaaatccagctggtccaatcgggagctgaggtcaagaagccgggagccaccgtcaagatct scFvcatgcaaggggtcgggattcaacatcgaggactactacattcactgggtgcagcaagctccg domain ntggaaaaggcctggaatggatgggcagaatcgacccagaaaacgacgaaactaagtacggaccgattttccaaggaagagtgactatcaccgccgatacttcaaccaataccgtctacatggaactgagctcgctccggtccgaagatactgcagtgtattactgtgcctttcgcggaggggtgtactggggccaaggaactactgtcactgtctcgtcaggaggcggagggtcgggaggaggcgggagcggaggcggtggctcgggtggcggaggaagcgacgtggtgatgacccagtccccggactccctcgccgtgagcctcggagagagggcgactatcaattgcaagtcgtcccagtcacttctggattccgatggtaaaacgtacctcaactggctgcagcaaaagccagggcagccacccaaacggttgatctcccttgtgtccaaactggatagcggagtgcctgaccgcttctcgggttccggtagcgggaccgacttcaccctgacgatcagctcactgcaggcggaggacgtggcagtgtactactgctggcagggaacccacttccctggcacctttggaggtggcaccaaggtggagatcaag CAR1 40atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleaaatccagctggtccaatcgggagctgaggtcaagaagccgggagccaccgtcaagatctc scFv - ntatgcaaggggtcgggattcaacatcgaggactactacattcactgggtgcagcaagctccgggaaaaggcctggaatggatgggcagaatcgacccagaaaacgacgaaactaagtacggaccgattttccaaggaagagtgactatcaccgccgatacttcaaccaataccgtctacatggaactgagctcgctccggtccgaagatactgcagtgtattactgtgcctttcgcggaggggtgtactggggccaaggaactactgtcactgtctcgtcaggaggcggagggtcgggaggaggcgggagcggaggcggtggctcgggtggcggaggaagcgacgtggtgatgacccagtccccggactccctcgccgtgagcctcggagagagggcgactatcaattgcaagtcgtcccagtcacttctggattccgatggtaaaacgtacctcaactggctgcagcaaaagccagggcagccacccaaacggttgatctcccttgtgtccaaactggatagcggagtgcctgaccgcttctcgggttccggtagcgggaccgacttcaccctgacgatcagctcactgcaggcggaggacgtggcagtgtactactgctggcagggaacccacttccctggcacctttggaggtggcaccaaggtggagatcaagggatcgcaccaccatcaccatcatcatcac CAR1 41Malpvtalllplalllhaarpeiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqap Solublegkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvy scFv - aawgqgttvtvssggggsggggsggggsggggsdvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikgshhhhhhhh CAR 1 - 42atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt agatccagctggtgcagtcgggagctgaagtcaaaaagcctggcgcaaccgtcaagatctcglentivirustgcaaaggatcagggttcaacatcgaggactactacatccattgggtgcaacaggcacccggaaaaggcctggagtggatggggaggattgacccagaaaatgacgaaaccaagtacggaccgatcttccaaggacgggtgaccatcacggctgacacttccactaacaccgtctacatggaactctcgagccttcgctcggaagataccgcggtgtactactgcgcctttagaggtggagtctactggggacaagggactaccgtcaccgtgtcgtcaggtggcggaggatcaggcggaggcggctccggtggaggaggaagcggaggaggtggctccgacgtggtgatgacgcagtcaccggactccttggcggtgagcctgggtgaacgcgccactatcaactgcaagagctcccagagcttgctggactccgatggaaagacttatctcaattggctgcaacagaagcctggccagccgccaaagagactcatctcactggtgagcaagctggatagcggagtgccagatcggttttcgggatcgggctcaggcaccgacttcaccctgactatttcctccctccaagccgaggatgtggccgtctactactgttggcaggggactcacttcccggggaccttcggtggaggcactaaggtggagatcaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 1 - 43malpvtalllplalllhaarpeiqlvqsgaevkkpgatvkisckgsgfnie dyyih wvqqapFull - aa gkglewmg ridpendetkygpifqg rvtitadtstntvymelsslrsedtavyycafrgg v ywgqgttvtvssggggsggggsggggsggggsdvvmtqspdslavslgeratinc kssqslldsdgktyln wlqqkpgqppkrlis lvsklds gvpdrfsgsgsgtdftltisslqaedva vyycwqgthfpgt fgggtkveiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 2 CAR244 dvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsg scFvvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikggggsggggsg domaingggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvt vss CAR245 gatgtcgtgatgacccagtccccagactccctcgcagtgtccttgggagaacgggccaccatc scFvaactgcaaatcgagccagtcactgctggactcagacggaaagacctacctcaactggctgcadomain - ntgcagaagcctggccagccaccgaagcgcctgatctccctggtgtccaagctggactcgggcgtcccggacaggtttagcggtagcggctcgggaaccgacttcactctgaccattagctcgctccaagctgaagatgtggcggtctactactgctggcaggggacccacttccccgggacctttggcggaggaactaaagtcgaaatcaaaggaggaggcggatcaggtggaggaggcagcggaggaggagggagcggcggtggcggctccgaaattcaacttgtgcaatccggtgccgaggtgaagaaacctggtgccactgtcaagatctcgtgtaagggatcgggattcaatatcgaggactactacatccactgggtgcaacaggcgccaggaaagggattggagtggatgggtcgcatcgacccggaaaacgatgagactaagtacggaccgatcttccaaggccgggtcacgatcactgcggatacctccactaataccgtgtatatggagctctcgtcactgagaagcgaagatacggccgtgtactactgcgcattcagaggaggtgtgtactggggccagggaactactgtgaccgtgtcgtcg CAR2 - 46atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg SolubleatgtcgtgatgacccagtccccagactccctcgcagtgtccttgggagaacgggccaccatcascFv - nt actgcaaatcgagccagtcactgctggactcagacggaaagacctacctcaactggctgcagcagaagcctggccagccaccgaagcgcctgatctccctggtgtccaagctggactcgggcgtcccggacaggtttagcggtagcggctcgggaaccgacttcactctgaccattagctcgctccaagctgaagatgtggcggtctactactgctggcaggggacccacttccccgggacctttggcggaggaactaaagtcgaaatcaaaggaggaggcggatcaggtggaggaggcagcggaggaggagggagcggcggtggcggctccgaaattcaacttgtgcaatccggtgccgaggtgaagaaacctggtgccactgtcaagatctcgtgtaagggatcgggattcaatatcgaggactactacatccactgggtgcaacaggcgccaggaaagggattggagtggatgggtcgcatcgacccggaaaacgatgagactaagtacggaccgatcttccaaggccgggtcacgatcactgcggatacctccactaataccgtgtatatggagctctcgtcactgagaagcgaagatacggccgtgtactactgcgcattcagaggaggtgtgtactggggccagggaactactgtgaccgtgtcgtcggggtcacatcaccaccatcatcatcaccac CAR2 - 47malpvtalllplalllhaarpdvvmtqspdslavslgeratinckssqslldsdgktylnwlqq SolublekpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggscFv - aa tkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssgshhhhhhhh CAR 2 - 48atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - ntacgtggtcatgactcaaagcccagattccttggctgtctcccttggagaaagagcaacgatcaattgcaaaagctcgcagtccctgttggactccgatggaaaaacctacctcaactggctgcagcagaagccgggacaaccaccaaagcggctgatttccctcgtgtccaagctggacagcggcgtgccggatcgcttctcgggcagcggctcgggaaccgattttactctcactatttcgtcactgcaagcggaggacgtggcggtgtattactgctggcagggcactcacttcccgggtacttttggtggaggtaccaaagtcgaaatcaagggtggaggcgggagcggaggaggcgggtcgggaggaggaggatcgggtggcggaggctcagaaatccagctggtgcagtcaggtgccgaagtgaagaagcctggggccacggtgaagatctcgtgcaaggggagcggattcaacatcgaggattactacatccattgggtgcaacaggcccctggcaaagggctggaatggatgggaaggatcgaccccgagaatgacgagactaagtacggcccgatcttccaaggacgggtgaccatcactgcagacacttcaaccaacaccgtctacatggaactctcctcgctgcgctccgaggacaccgccgtgtactactgtgctttcagaggaggagtctactggggacagggaacgaccgtgaccgtcagctcaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 2 - 49malpvtalllplalllhaarpdvvmtqspdslavslgeratinc kssqslldsdgktyln wlqFull - aa qkpgqppkrlis lvsklds gvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgt fg ggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyih wvqqapgkglewmg ridpendetkygpifqg rvtitadtstntvymelsslrse d tavyycafrggvy wgqgttvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 3 CAR350 eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetk scFvygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssggggsg domaingggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkv eik CAR351 gagattcagctggtccaaagcggcgcagaagtgaaaaagccaggggaatcgttgcgcatca scFvgctgtaaaggttccggcttcaacatcgaggactattacatccattgggtgcggcagatgccagdomain nt gaaaggggctggaatggatgggacggattgacccggagaacgacgaaaccaagtacggaccgatctttcaaggacacgtgactatctccgccgacaccagcatcaatacggtgtacctccaatggtcctcactcaaggcctcggataccgcgatgtactactgcgcgttcagaggaggcgtctactggggacaagggactactgtgactgtctcatcaggaggtggaggaagcggaggaggtggctcgggcggaggtggatcgggaggaggagggtccgatgtggtgatgacccagtccccactgtcgctcccggtgaccctcggacagcctgctagcatctcgtgcaaatcctcgcaatccctgctggactcggacggaaaaacgtacctcaattggctgcagcagcgccctggccagagcccgagaaggcttatctcgctggtgtcaaagctggatagcggtgtgcccgaccggttcagcggctcagggtcaggaaccgatttcaccttgaagatctcccgcgtggaagccgaagatgtcggagtctactactgctggcagggtactcacttcccggggacctttggtggcggcactaaggtcgagattaag CAR 3 - 52atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleagattcagctggtccaaagcggcgcagaagtgaaaaagccaggggaatcgttgcgcatcag scFv - ntctgtaaaggttccggcttcaacatcgaggactattacatccattgggtgcggcagatgccaggaaaggggctggaatggatgggacggattgacccggagaacgacgaaaccaagtacggaccgatctttcaaggacacgtgactatctccgccgacaccagcatcaatacggtgtacctccaatggtcctcactcaaggcctcggataccgcgatgtactactgcgcgttcagaggaggcgtctactggggacaagggactactgtgactgtctcatcaggaggtggaggaagcggaggaggtggctcgggcggaggtggatcgggaggaggagggtccgatgtggtgatgacccagtccccactgtcgctcccggtgaccctcggacagcctgctagcatctcgtgcaaatcctcgcaatccctgctggactcggacggaaaaacgtacctcaattggctgcagcagcgccctggccagagcccgagaaggcttatctcgctggtgtcaaagctggatagcggtgtgcccgaccggttcagcggctcagggtcaggaaccgatttcaccttgaagatctcccgcgtggaagccgaagatgtcggagtctactactgctggcagggtactcacttcccggggacctttggtggcggcactaaggtcgagattaagggctcacaccatcatcaccatcaccaccac CAR 3 - 53malpvtalllplalllhaarpeiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmp Solublegkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrgg scFv - aavywgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikgshhhhhhhh CAR 3 - 54atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt aaatccagctggtgcaaagcggagccgaggtgaagaagcccggagaatccctgcgcatctcgtgtaagggttccggctttaacatcgaggattactacatccactgggtgagacagatgccgggcaaaggtctggaatggatgggccgcatcgacccggagaacgacgaaaccaaatacggaccaatcttccaaggacatgtgactatttccgcggatacctccatcaacactgtctacttgcagtggagctcgctcaaggcgtcggataccgccatgtactactgcgcattcagaggaggtgtgtactggggccagggcactacggtcaccgtgtcctcgggaggtggagggtcaggaggcggaggctcgggcggtggaggatcaggcggaggaggaagcgatgtggtcatgactcaatccccactgtcactgcctgtcactctggggcaaccggcttccatctcatgcaagtcaagccaatcgctgctcgactccgacggaaaaacctacctcaattggcttcagcagcgcccaggccagtcgcctcggaggctgatctcactcgtgtcgaagcttgactccggggtgccggatcggtttagcggaagcggatcggggaccgacttcacgttgaagattagccgggtggaagccgaggacgtgggagtctattactgctggcaggggacccacttcccggggactttcggaggaggcaccaaagtcgagattaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 3 - 55malpvtalllplalllhaarpeiqlvqsgaevkkpgeslrisckgsgfnie dyyih wvrqmp Full-aagkglewmg ridpendetkygpifqg hvtisadtsintvylqwsslkasdtamyycaf rg gvywgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisc ks sqslldsdgktylnwlqqrpgqsprrlis lvsklds gvpdrfsgsgsgtdftlkisrveaedv gvyyc wqgthfpgtfgggtkveiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr CAR 4CAR4 56 dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvscFv pdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikggggsggggsg domaingggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttv tvss CAR457 gacgtcgtcatgacccagagcccgctgtcactgcctgtgaccctgggccagccggcgtccat scFvtagctgcaaatcctcgcaatccctgctcgactcagacggaaaaacgtacttgaactggctccaadomain ntcagcgccctgggcaatccccaaggcggcttatctcactcgtcagcaagctcgatagcggtgtcccagacagattttcgggctcgggatcgggcactgatttcactctgaagatctcgcgggtggaagccgaggatgtgggagtgtactattgctggcagggcactcacttccccgggacgtttggcggaggaactaaggtcgagatcaaaggaggaggtggatcaggcggaggtgggagcggaggaggaggaagcggtggtggaggttccgaaatccagctggtgcaatcaggagccgaggtgaagaagccgggagaatccctgcgcatctcgtgcaagggctcgggcttcaacatcgaggattactacatccactgggtgcggcagatgccgggaaaggggttggaatggatgggacgcattgacccggaaaatgatgaaaccaaatacgggccaatcttccaaggccacgtgaccattagcgctgacacttccatcaacaccgtgtaccttcagtggtcctcactgaaggcgtcggacactgccatgtactactgtgcattcagaggaggggtctactggggacagggcaccaccgtgaccgtgagctcc CAR4 - 58atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg SolubleacgtcgtcatgacccagagcccgctgtcactgcctgtgaccctgggccagccggcgtccattascFv - ntgctgcaaatcctcgcaatccctgctcgactcagacggaaaaacgtacttgaactggctccaacagcgccctgggcaatccccaaggcggcttatctcactcgtcagcaagctcgatagcggtgtcccagacagattttcgggctcgggatcgggcactgatttcactctgaagatctcgcgggtggaagccgaggatgtgggagtgtactattgctggcagggcactcacttccccgggacgtttggcggaggaactaaggtcgagatcaaaggaggaggtggatcaggcggaggtgggagcggaggaggaggaagcggtggtggaggttccgaaatccagctggtgcaatcaggagccgaggtgaagaagccgggagaatccctgcgcatctcgtgcaagggctcgggcttcaacatcgaggattactacatccactgggtgcggcagatgccgggaaaggggttggaatggatgggacgcattgacccggaaaatgatgaaaccaaatacgggccaatcttccaaggccacgtgaccattagcgctgacacttccatcaacaccgtgtaccttcagtggtcctcactgaaggcgtcggacactgccatgtactactgtgcattcagaggaggggtctactggggacagggcaccaccgtgaccgtgagctccggctcgcatcaccatcatcaccaccatcac CAR4 - 59malpvtalllplalllhaarpdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqq SolublerpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggscFv - aa tkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssgshhhhhhhh CAR 4 - 60atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - ntacgtcgtcatgacccaatcccctctctccctgccggtcaccctgggtcagccggcgtcgatctcatgcaaaagctcacagtccctgctggattcggacggaaaaacctacttgaactggctccaacagaggccgggtcagtcccctcgcagactgatctcgctggtgagcaagctcgactcgggtgtgccggatcggttctccgggtcaggatcgggcaccgactttacgctcaagatttcgagagtggaggccgaggatgtgggagtgtactattgctggcagggcacgcatttccccgggacctttggaggcgggactaaggtggaaatcaagggaggtggcggatcaggcggaggaggcagcggcggaggtggatcaggaggcggagggtcagagatccagctggtccaaagcggagcagaggtgaagaagccaggcgagtcccttcgcatttcgtgcaaagggagcggcttcaacattgaagattactacatccactgggtgcggcaaatgccaggaaagggtctggaatggatgggacggatcgacccagaaaatgatgaaactaagtacggaccgatcttccaaggacacgtcactatctccgcggacacttcgatcaacaccgtgtacctccagtggagcagcttgaaagcctccgacaccgctatgtactactgtgccttccgcggaggagtctactggggacaggggactactgtgaccgtgtcgtccaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 4 - 61malpvtalllplalllhaarpdvvmtqsplslpvtlgqpasisc kssqslldsdgktyln wlqFull - aa qrpgqsprrlis lvsklds gvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgt fgggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmg ridpendetkygpifqg hvtisadtsintvylqwsslkasd tamyycafrggvy wgqgttvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr CAR 5CAR5 62 eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendet scFvkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssggggsg domaingggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkv eik CAR563 gaaatccagctcgtgcagagcggagccgaggtcaagaaaccgggtgctaccgtgaagattt scFvcatgcaagggatcgggcttcaacatcgaggattactacatccactgggtgcagcaggcacca domain ntggaaaaggacttgaatggatgggccggatcgacccggaaaatgacgagactaagtacggccctatcttccaaggacgggtgacgatcaccgcagacactagcaccaacaccgtctatatggaactctcgtccctgaggtccgaagatactgccgtgtactactgtgcgtttcgcggaggtgtgtactggggacagggtaccaccgtcaccgtgtcatcgggcggtggaggctccggtggaggagggtcaggaggcggtggaagcggaggaggcggcagcgacgtggtcatgactcaatcgccgctgtcgctgcccgtcactctgggacaacccgcgtccatcagctgcaaatcctcgcagtcactgcttgactccgatggaaagacctacctcaactggctgcagcaacgcccaggccaatccccaagacgcctgatctcgttggtgtcaaagctggactcaggggtgccggaccggttctccgggagcgggtcgggcacggatttcactctcaagatctccagagtggaagccgaggatgtgggagtctactactgctggcagggaacccatttccctggaacttttggcggaggaactaaggtcgagattaaa CAR5- 64atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleaaatccagctcgtgcagagcggagccgaggtcaagaaaccgggtgctaccgtgaagatttca scFv-nttgcaagggatcgggcttcaacatcgaggattactacatccactgggtgcagcaggcaccaggaaaaggacttgaatggatgggccggatcgacccggaaaatgacgagactaagtacggccctatcttccaaggacgggtgacgatcaccgcagacactagcaccaacaccgtctatatggaactctcgtccctgaggtccgaagatactgccgtgtactactgtgcgtttcgcggaggtgtgtactggggacagggtaccaccgtcaccgtgtcatcgggcggtggaggctccggtggaggagggtcaggaggcggtggaagcggaggaggcggcagcgacgtggtcatgactcaatcgccgctgtcgctgcccgtcactctgggacaacccgcgtccatcagctgcaaatcctcgcagtcactgcttgactccgatggaaagacctacctcaactggctgcagcaacgcccaggccaatccccaagacgcctgatctcgttggtgtcaaagctggactcaggggtgccggaccggttctccgggagcgggtcgggcacggatttcactctcaagatctccagagtggaagccgaggatgtgggagtctactactgctggcagggaacccatttccctggaacttttggcggaggaactaaggtcgagattaaagggagccaccatcatcatcaccaccaccac CAR5 - 65malpvtalllplalllhaarpeiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqap Solublegkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvy scFv - aawgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikgshhhhhhhh CAR 5 - 66atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt aaatccagctcgtgcagagcggagccgaggtcaagaaaccgggtgctaccgtgaagatttcatgcaagggatcgggcttcaacatcgaggattactacatccactgggtgcagcaggcaccaggaaaaggacttgaatggatgggccggatcgacccggaaaatgacgagactaagtacggccctatcttccaaggacgggtgacgatcaccgcagacactagcaccaacaccgtctatatggaactctcgtccctgaggtccgaagatactgccgtgtactactgtgcgtttcgcggaggtgtgtactggggacagggtaccaccgtcaccgtgtcatcgggcggtggaggctccggtggaggagggtcaggaggcggtggaagcggaggaggcggcagcgacgtggtcatgactcaatcgccgctgtcgctgcccgtcactctgggacaacccgcgtccatcagctgcaaatcctcgcagtcactgcttgactccgatggaaagacctacctcaactggctgcagcaacgcccaggccaatccccaagacgcctgatctcgttggtgtcaaagctggactcaggggtgccggaccggttctccgggagcgggtcgggcacggatttcactctcaagatctccagagtggaagccgaggatgtgggagtctactactgctggcagggaacccatttccctggaacttttggcggaggaactaaggtcgagattaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 5- 67malpvtalllplalllhaarpeiqlvqssaevkkpsatvkiscksssfnie dyyih wvqqap Full-aagkglewmg ridpendetkygpifqg rvtitadtstntvymelsslrsedtavyycaf rgg vywgqgttvtvssggggsggggsggggsggggsdvvmtqsplslpvtlgqpasisc kss qslldsdgktylnwlqqrpgqsprrlis lvsklds gvpdrfsgsgsgtdftlkisrveaedvg vyyc wqgthfpgtfgggtkveiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 6 CAR668 eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetk scFvygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssggggsg domaingggsggggsggggsdvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkv eik CAR669 gaaatccagctggtgcagtcaggcgccgaggtcaagaagccgggagagtcgctgagaatct scFvcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccg domain ntggaaagggactggaatggatgggccggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctcgcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcgggaggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgaccatcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtcaaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgttggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaag CAR6 - 70atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleaaatccagctggtgcagtcaggcgccgaggtcaagaagccgggagagtcgctgagaatctc scFv - ntgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggccggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctcgcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcgggaggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgaccatcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtcaaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgttggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatcacaccaccatcatcaccatcaccaccat CAR6 - 71malpvtalllplalllhaarpeiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmp Solublegkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrgg scFv - aavywgqgttvtvssggggsggggsggggsggggsdvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikgshhhhhhhhh CAR6 - 72atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt agattcagctcgtgcaatcgggagcggaagtcaagaagccaggagagtccttgcggatctcatgcaagggtagcggctttaacatcgaggattactacatccactgggtgaggcagatgccggggaagggactcgaatggatgggacggatcgacccagaaaacgacgaaactaagtacggtccgatcttccaaggccatgtgactattagcgccgatacttcaatcaataccgtgtatctgcaatggtcctcattgaaagcctcagataccgcgatgtactactgtgctttcagaggaggggtctactggggacagggaactaccgtgactgtctcgtccggcggaggcgggtcaggaggtggcggcagcggaggaggagggtccggcggaggtgggtccgacgtcgtgatgacccagagccctgacagcctggcagtgagcctgggcgaaagagctaccattaactgcaaatcgtcgcagagcctgctggactcggacggaaaaacgtacctcaattggctgcagcaaaagcctggccagccaccgaagcgccttatctcactggtgtcgaagctggattcgggagtgcccgatcgcttctccggctcgggatcgggtactgacttcaccctcactatctcctcgcttcaagcagaggacgtggccgtctactactgctggcagggaacccactttccgggaaccttcggcggagggacgaaagtggagatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR6 - 73malpvtalllplalllhaarpeiqlvqsgaevkkpgeslrisckgsgfnie dyyih wvrqmpFull - aa gkglewmg ridpendetkygpifqg hvtisadtsintvylqwsslkasdtamyycaf rggvy wgqgttvtvssggggsggggsggggsggggsdvvmtqspdslavslgeratinc kssqslldsdgktyln wlqqkpgqppkrlis lvsklds gvpdrfsgsgsgtdftltisslqaedv avyycwqgthfpgt fgggtkveiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr CAR 7CAR7 74 dvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvskldsgscFv vpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggtkveikggggsggggsgdomain gggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttv tvss CAR775 gacgtggtgatgacccaatcgccagattccctggcagtgtccctgggcgaacgcgccactatt scFvaactgcaaatcgtcacagtccttgcttgattccgacggaaagacctacctcaattggctccagcdomain nt agaagccaggacaaccgccaaagagactgatctccctggtgtcaaagctggactcgggagtgcctgatcggttctcgggtagcgggagcggcaccgacttcactctgaccatctcgtcactccaggctgaggacgtggccgtgtattactgttggcagggtactcactttccgggcactttcggaggcggcaccaaggtggagattaaaggaggaggcggaagcggaggtggaggatcgggaggtggtgggagcggcggaggagggagcgagatccagctcgtccaatcgggagcggaagtgaagaagcccggagagtcacttagaatctcatgcaaggggtcgggcttcaacatcgaggattactacatccattgggtccgccagatgcctggtaaaggactggaatggatggggaggattgacccggaaaacgacgaaactaagtacggaccgatctttcaagggcacgtgactatctccgctgatacctcaatcaatactgtctacctccagtggtcctcgctgaaagcaagcgacaccgcgatgtactactgcgccttccggggaggagtgtactggggccaaggcaccacggtcacggtcagctcc CAR7 - 76atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg SolubleacgtggtgatgacccaatcgccagattccctggcagtgtccctgggcgaacgcgccactattascFv - ntactgcaaatcgtcacagtccttgcttgattccgacggaaagacctacctcaattggctccagcagaagccaggacaaccgccaaagagactgatctccctggtgtcaaagctggactcgggagtgcctgatcggttctcgggtagcgggagcggcaccgacttcactctgaccatctcgtcactccaggctgaggacgtggccgtgtattactgttggcagggtactcactttccgggcactttcggaggcggcaccaaggtggagattaaaggaggaggcggaagcggaggtggaggatcgggaggtggtgggagcggcggaggagggagcgagatccagctcgtccaatcgggagcggaagtgaagaagcccggagagtcacttagaatctcatgcaaggggtcgggcttcaacatcgaggattactacatccattgggtccgccagatgcctggtaaaggactggaatggatggggaggattgacccggaaaacgacgaaactaagtacggaccgatctttcaagggcacgtgactatctccgctgatacctcaatcaatactgtctacctccagtggtcctcgctgaaagcaagcgacaccgcgatgtactactgcgccttccggggaggagtgtactggggccaaggcaccacggtcacggtcagctccggctcccatcaccaccaccatcaccatcatcac CAR7 - 77malpvtalllplalllhaarpdvvmtqspdslavslgeratinckssqslldsdgktylnwlqq SolublekpgqppkrlislvskldsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfgggscFv - aa tkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetkygpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgttvtvssgshhhhhhhhh CAR 7 78atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - ntacgtggtgatgactcagtcgcctgactcgctggctgtgtcccttggagagcgggccactatcaattgcaagtcatcccagtcgctgctggattccgacgggaaaacctacctcaattggctgcagcaaaaaccgggacagcctccaaagcggctcatcagcctggtgtccaagttggacagcggcgtgccagaccgcttctccggttcgggaagcggtactgatttcacgctgaccatctcatccctccaagcggaggatgtggcagtctactactgttggcagggcacgcattttccgggcacttttggaggagggaccaaggtcgaaatcaagggaggaggtggctcgggcggaggaggctcgggaggaggaggatcaggaggcggtggaagcgagattcaactggtccagagcggcgcagaagtcaagaagccgggtgaatcgctcagaatctcgtgcaaaggatcgggattcaacatcgaggactactacattcactgggtcagacaaatgccgggcaaagggctggaatggatggggaggatcgaccccgaaaacgatgaaaccaagtacggaccaatcttccaagggcacgtgaccatttcggcggacacctcaatcaacactgtgtacctccagtggagctcacttaaggccagcgataccgccatgtactattgcgctttccgcggaggggtgtactggggacagggcactactgtgaccgtgtcatccaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 7 79malpvtalllplalllhaarpdvvmtqspdslavslgeratinc kssqslldsdgktyln wlqFull - aa qkpgqppkrlis lvsklds gvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgt fg ggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgeslrisckgsgfniedy yih wvrqmpgkglewmg ridpendetkygpifqg hvtisadtsintvylqwsslkasdtamyycaf rggvy wgqgttvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr CAR 8CAR8 80 dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvskldsgvscFv pdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggtkveikggggsggggsg domaingggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvt vss CAR881 gatgtggtcatgacgcagtcaccactgtccctccccgtgacccttggacagccagcgtcgatt scFvagctgcaagtcatcccaatccctgctcgattcggatggaaagacctatctcaactggctgcagcdomain nt aaagacccggtcagagccctaggagactcatctcgttggtgtcaaagctggacagcggagtgccggaccggttttccggttcgggatcggggacggacttcactctgaagatttcacgggtggaagctgaggatgtgggagtgtactactgctggcagggaacccatttccctggcacttttggcggaggaactaaggtcgaaatcaagggaggaggtggctcgggaggaggcggatcgggcggaggcgggagcggcggaggagggtccgaaatccaacttgtccagtcaggagccgaagtgaagaaaccgggagccaccgtcaaaatcagctgtaagggatcgggattcaatatcgaggactactacatccactgggtgcagcaagctccgggcaaaggactggagtggatggggcgcatcgacccagagaacgacgaaaccaaatacggcccgatcttccaagggcgggtgaccatcaccgcggacacctcaactaacactgtgtacatggagctgagctccctgcgctccgaagatactgcagtctactactgcgccttccgcggtggtgtgtactggggacagggcaccactgtgactgtcagctcg CAR8 - 82atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg SolubleatgtggtcatgacgcagtcaccactgtccctccccgtgacccttggacagccagcgtcgattascFv - ntgctgcaagtcatcccaatccctgctcgattcggatggaaagacctatctcaactggctgcagcaaagacccggtcagagccctaggagactcatctcgttggtgtcaaagctggacagcggagtgccggaccggttttccggttcgggatcggggacggacttcactctgaagatttcacgggtggaagctgaggatgtgggagtgtactactgctggcagggaacccatttccctggcacttttggcggaggaactaaggtcgaaatcaagggaggaggtggctcgggaggaggcggatcgggcggaggcgggagcggcggaggagggtccgaaatccaacttgtccagtcaggagccgaagtgaagaaaccgggagccaccgtcaaaatcagctgtaagggatcgggattcaatatcgaggactactacatccactgggtgcagcaagctccgggcaaaggactggagtggatggggcgcatcgacccagagaacgacgaaaccaaatacggcccgatcttccaagggcgggtgaccatcaccgcggacacctcaactaacactgtgtacatggagctgagctccctgcgctccgaagatactgcagtctactactgcgccttccgcggtggtgtgtactggggacagggcaccactgtgactgtcagctcggggtcccaccatcatcaccaccaccatcac CAR8 - 83malpvtalllplalllhaarpdvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqq SolublerpgqsprrlislvskldsgvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgtfgggscFv - aa tkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetkygpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgttvtvssgshhhhhhhh CAR 8 - 84atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - ntatgtggtcatgacgcagtcaccactgtccctccccgtgacccttggacagccagcgtcgattagctgcaagtcatcccaatccctgctcgattcggatggaaagacctatctcaactggctgcagcaaagacccggtcagagccctaggagactcatctcgttggtgtcaaagctggacagcggagtgccggaccggttttccggttcgggatcggggacggacttcactctgaagatttcacgggtggaagctgaggatgtgggagtgtactactgctggcagggaacccatttccctggcacttttggcggaggaactaaggtcgaaatcaagggaggaggtggctcgggaggaggcggatcgggcggaggcgggagcggcggaggagggtccgaaatccaacttgtccagtcaggagccgaagtgaagaaaccgggagccaccgtcaaaatcagctgtaagggatcgggattcaatatcgaggactactacatccactgggtgcagcaagctccgggcaaaggactggagtggatggggcgcatcgacccagagaacgacgaaaccaaatacggcccgatcttccaagggcgggtgaccatcaccgcggacacctcaactaacactgtgtacatggagctgagctccctgcgctccgaagatactgcagtctactactgcgccttccgcggtggtgtgtactggggacagggcaccactgtgactgtcagctcgaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 8 - 85malpvtalllplalllhaarpdvvmtqsplslpvtlgqpasisc ksscislldsdgktyln wlqFull - aa qrpgqsprrlis lvsklds gvpdrfsgsgsgtdftlkisrveaedvgvyycwqgthfpgt fg ggtkveikggggsggggsggggsggggseiqlvqsgaevkkpgatvkisckgsgfnied yyih wvqqapgkglewmg ridpendetkygpifqg rvtitadtstntvymelsslrsedtavyycaf rggvy wgqgttvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalpprCAR 9 Mouse anti-EGFRvIII clone 3C10 CAR9 86eiqlqqsgaelvkpgasvklsctgsgfniedyyihwvkqrteqglewigridpendetkyg scFvpifqgratitadtssntvylqlssltsedtavyycafrggvywgpgttltvssggggsggggsg domaingggshmdvvmtqspltlsvaigqsasisckssqslldsdgktylnwllqrpgqspkrlislvskldsgvpdrftgsgsgtdftlrisrveaedlgiyycwqgthfpgtfgggtkleik CAR9 98gagatccagctccaacagagcggagccgaactggtcaaaccgggagcgtcggtgaagttgt scFvcatgcactggatcgggcttcaacatcgaggattactacatccactgggtcaagcaacgcaccgdomain nt agcaggggctggaatggatcggacggatcgaccccgaaaacgatgaaaccaagtacgggcctatcttccaaggacgggccaccattacggctgacacgtcaagcaataccgtctacctccagctttccagcctgacctccgaggacactgccgtgtactactgcgccttcagaggaggcgtgtactggggaccaggaaccactttgaccgtgtccagcggaggcggtggatcaggaggaggaggctcaggcggtggcggctcgcacatggacgtggtcatgactcagtccccgctgaccctgtcggtggcaattggacagagcgcatccatctcgtgcaagagctcacagtcgctgctggattccgacggaaagacttatctgaactggctgctccaaagaccagggcaatcaccgaaacgccttatctccctggtgtcgaaactcgactcgggtgtgccggatcggtttaccggtagcgggtccggcacggacttcactctccgcatttcgagggtggaagcggaggatctcgggatctactactgttggcagggaacccacttccctgggacttttggaggcggaactaagctggaaatcaag CAR9 - 87atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleagatccagctccaacagagcggagccgaactggtcaaaccgggagcgtcggtgaagttgtc scFv - ntatgcactggatcgggcttcaacatcgaggattactacatccactgggtcaagcaacgcaccgagcaggggctggaatggatcggacggatcgaccccgaaaacgatgaaaccaagtacgggcctatcttccaaggacgggccaccattacggctgacacgtcaagcaataccgtctacctccagctttccagcctgacctccgaggacactgccgtgtactactgcgccttcagaggaggcgtgtactggggaccaggaaccactttgaccgtgtccagcggaggcggtggatcaggaggaggaggctcaggcggtggcggctcgcacatggacgtggtcatgactcagtccccgctgaccctgtcggtggcaattggacagagcgcatccatctcgtgcaagagctcacagtcgctgctggattccgacggaaagacttatctgaactggctgctccaaagaccagggcaatcaccgaaacgccttatctccctggtgtcgaaactcgactcgggtgtgccggatcggtttaccggtagcgggtccggcacggacttcactctccgcatttcgagggtggaagcggaggatctcgggatctactactgttggcagggaacccacttccctgggacttttggaggcggaactaagctggaaatcaagggtagccatcaccatcaccaccaccatcat CAR9 - 88malpvtalllplalllhaarpeiqlqqsgaelvkpgasvklsctgsgfniedyyihwvkqrte SolubleqglewigridpendetkygpifqgratitadtssntvylqlssltsedtavyycafrggvywgscFv - aa pgttltvssggggsggggsggggshmdvvmtqspltlsvaigqsasisckssqslldsdgktylnwllqrpgqspkrlislvskldsgvpdrftgsgsgtdftlrisrveaedlgiyycwqgthfpgtfgggtkleikgshhhhhhhh CAR 9 - 89atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt agatccagctccaacagagcggagccgaactggtcaaaccgggagcgtcggtgaagttgtcatgcactggatcgggcttcaacatcgaggattactacatccactgggtcaagcaacgcaccgagcaggggctggaatggatcggacggatcgaccccgaaaacgatgaaaccaagtacgggcctatcttccaaggacgggccaccattacggctgacacgtcaagcaataccgtctacctccagctttccagcctgacctccgaggacactgccgtgtactactgcgccttcagaggaggcgtgtactggggaccaggaaccactttgaccgtgtccagcggaggcggtggatcaggaggaggaggctcaggcggtggcggctcgcacatggacgtggtcatgactcagtccccgctgaccctgtcggtggcaattggacagagcgcatccatctcgtgcaagagctcacagtcgctgctggattccgacggaaagacttatctgaactggctgctccaaagaccagggcaatcaccgaaacgccttatctccctggtgtcgaaactcgactcgggtgtgccggatcggtttaccggtagcgggtccggcacggacttcactctccgcatttcgagggtggaagcggaggatctcgggatctactactgttggcagggaacccacttccctgggacttttggaggcggaactaagctggaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 9- 90malpvtalllplalllhaarpeiqlqqsgaelvkpgasvklsctgsgfnie dyyih wvkqrteFull-aa qglewig ridpendetkygpifqg ratitadtssntvylqlssltsedtavyyca frggvyw gpgttltvssggggsggggsggggshmdvvmtqspltlsvaigqsasisc kssqslldsdg ktylnwllqrpgqspkrlis lvsklds gvpdrftgsgsgtdftlrisrveaedlgiyyc wqgt hfpgtfgggtklei0tlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalpprCAR10 Anti-EGFRvIII clone 139 CAR10 91diqmtqspsslsasvgdrvtitcrasqgirnnlawyqqkpgkapkrliyaasnlqsgvpsrft scFvgsgsgteftlivsslqpedfatyyclqhhsypltsgggtkveikrtgstsgsgkpgsgegsev domainqvlesggglvqpggslrlscaasgftfssyamswvrqapgkglewvsaisgsggstnyadsvkgrftisrdnskntlylqmnslraedtavyycagssgwseywgqgtlvtvss CAR9 92gatatccaaatgactcagagcccttcatccctgagcgccagcgtcggagacagggtgaccat scFvcacgtgccgggcatcccaaggcattagaaataacttggcgtggtatcagcaaaaaccaggaa domain ntaggccccgaagcgcctgatctacgcggcctccaaccttcagtcaggagtgccctcgcgcttcaccgggagcggtagcggaactgagtttacccttatcgtgtcgtccctgcagccagaggacttcgcgacctactactgcctccagcatcactcgtacccgttgacttcgggaggcggaaccaaggtcgaaatcaaacgcactggctcgacgtcagggtccggtaaaccgggatcgggagaaggatcggaagtccaagtgctggagagcggaggcggactcgtgcaacctggcgggtcgctgcggctcagctgtgccgcgtcgggttttactttcagctcgtacgctatgtcatgggtgcggcaggctccgggaaaggggctggaatgggtgtccgctatttccggctcgggtggaagcaccaattacgccgactccgtgaagggacgcttcaccatctcacgggataactccaagaatactctgtacctccagatgaactcgctgagagccgaggacaccgcagtgtactactgcgcagggtcaagcggctggtccgaatactggggacagggcaccctcgtcactgtcagctcc CAR10 - 93atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccg Solubleatatccaaatgactcagagcccttcatccctgagcgccagcgtcggagacagggtgaccatc scFv - ntacgtgccgggcatcccaaggcattagaaataacttggcgtggtatcagcaaaaaccaggaaaggccccgaagcgcctgatctacgcggcctccaaccttcagtcaggagtgccctcgcgcttcaccgggagcggtagcggaactgagtttacccttatcgtgtcgtccctgcagccagaggacttcgcgacctactactgcctccagcatcactcgtacccgttgacttcgggaggcggaaccaaggtcgaaatcaaacgcactggctcgacgtcagggtccggtaaaccgggatcgggagaaggatcggaagtccaagtgctggagagcggaggcggactcgtgcaacctggcgggtcgctgcggctcagctgtgccgcgtcgggttttactttcagctcgtacgctatgtcatgggtgcggcaggctccgggaaaggggctggaatgggtgtccgctatttccggctcgggtggaagcaccaattacgccgactccgtgaagggacgcttcaccatctcacgggataactccaagaatactctgtacctccagatgaactcgctgagagccgaggacaccgcagtgtactactgcgcagggtcaagcggctggtccgaatactggggacagggcaccctcgtcactgtcagctcccatcaccatcaccaccaccatcac CAR10 - 94malpvtalllplalllhaarpdiqmtqspsslsasvgdrvtitcrasqgirnnlawyqqkpgk SolubleapkrliyaasnlqsgvpsrftgsgsgteftlivsslqpedfatyyclqhhsypltsgggtkveikscFv-aa rtgstsgsgkpgsgegsevqvlesggglvqpggslrlscaasgftfssyamswvrqapgkglewvsaisgsggstnyadsvkgrftisrdnskntlylqmnslraedtavyycagssgwseywgqgtlvtvsshhhhhhhh CAR 10 95atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgFull - nt atatccaaatgactcagagcccttcatccctgagcgccagcgtcggagacagggtgaccatcacgtgccgggcatcccaaggcattagaaataacttggcgtggtatcagcaaaaaccaggaaaggccccgaagcgcctgatctacgcggcctccaaccttcagtcaggagtgccctcgcgcttcaccgggagcggtagcggaactgagtttacccttatcgtgtcgtccctgcagccagaggacttcgcgacctactactgcctccagcatcactcgtacccgttgacttcgggaggcggaaccaaggtcgaaatcaaacgcactggctcgacgtcagggtccggtaaaccgggatcgggagaaggatcggaagtccaagtgctggagagcggaggcggactcgtgcaacctggcgggtcgctgcggctcagctgtgccgcgtcgggttttactttcagctcgtacgctatgtcatgggtgcggcaggctccgggaaaggggctggaatgggtgtccgctatttccggctcgggtggaagcaccaattacgccgactccgtgaagggacgcttcaccatctcacgggataactccaagaatactctgtacctccagatgaactcgctgagagccgaggacaccgcagtgtactactgcgcagggtcaagcggctggtccgaatactggggacagggcaccctcgtcactgtcagctccaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg CAR 10 96malpvtalllplalllhaarpdiqmtqspsslsasvgdrvtitcrasqgirnnlawyqqkpgkFull - aaapkrliyaasnlqsgvpsrftgsgsgteftlivsslqpedfatyyclqhhsypltsgggtkveikrtgstsgsgkpgsgegsevqvlesggglvqpggslrlscaasgftfssyamswvrqapgkglewvsaisgsggstnyadsvkgrftisrdnskntlylqmnslraedtavyycagssgwseywgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr

The CAR scFv fragments were then cloned into lentiviral vectors tocreate a full length CAR construct in a single coding frame, and usingthe EF1 alpha promoter for expression (SEP ID NO: 97).

EF1 alpha promoter (SEQ ID NO: 97)GTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA.Surface Expression of CAR9, CAR10 and Select Humanized EGFRvIII CARConstructs and Staining by FACS

The following experiments showed that there appears to be an affinitydifference for EGFRvIII based in vitro binding studies in both Jurkatcells and primary T cells.

Jurkat E6 cells were electroporated with either CAR9 vector or CAR10vector using Amaxa Cell Line Nucleofector Kit V (Lonza, Colgne AG,Germany) and program X-001. One day after the transfection, 0.5×10⁶cells were placed into each well of a V-shape 96 well plate (GreinerBio-One, Germany) in 0.2 ml FACS buffer (DPBS buffer containing 5% FBS)and incubated for 10 minutes at room temperature. Cells were then spundown and resuspended in 0.2 ml of the FACS buffer with differentconcentrations of EGFRvIII-Fc or EGFRwt-Fc and incubated at 4° C. for 30minutes. Cells were then washed with FACS buffer three times, andincubated with 0.2 ml of the FACS buffer with 2 μl of PE anti-human IgGFc (Jackson ImmunoResearch Laboratories, West Grove, Pa.) for 30 minutesat 4° C. in the dark. After washing with 0.2 ml of FACS buffer threetimes, cells were analyzed on a LSRII (BD Biosciences, San Jose, Calif.)machine using the FACSDiva software (BD Biosciences, San Jose, Calif.).Immunofluorescence staining was analyzed as the relative logfluorescence of live cells, and the percentage of the PE positive cellswere measured.

As shown in FIGS. 12A and 12B, binding of the CAR9 expressed in Jurkatcells to EGFRvIII-Fc fusion protein is approximately 1000 fold strongerthan to wild type EGFR-Fc. Furthermore, the CART construct expressingCAR10 exhibits a significantly lower (˜40 fold) binding to EGFRvIIIcompared to CAR9. This suggests that although murine CAR9 binds toEGFRvIII, it still retains some binding to wild type EGFR. Moreover, itstrongly indicates that CAR9 has a higher binding affinity for EGFRvIIIthan the CAR10 construct.

Further experiments in primary T cells yielded similar results. Briefly,primary human CD3+ T cells were stimulated with anti-CD3/CD28 beads for24 hrs and then transduced with lentiviral vectors encoding either CAR9,CAR10, CAR6 or a control CAR at a MOI of 3:1. Included in the experimentwas also a mock transduced T cell population. These cells were expandedfor about 8-9 days in culture until they started to rest down. At thispoint, 0.5×106 cells were placed into each well of a V-shape 96 wellplate. The cells were washed one time with PBS and stained withLive/Dead reagent (1:1000 in PBS) for 30 min on ice. Cells were thenwashed twice with FACS buffer and incubated with 1 μg/ml biotinylatedEGFRvIII or EGFR wt protein for 30 min on ice. Cells were then washedtwo times and incubated with 0.2 ml of FACS buffer with 1:1000 dilutionof streptavidin-PE for 15 min on ice. After washing twice with FACSbuffer, cells were analyzed on a LSRII. Immunofluorescence staining wasanalyzed as the relative log fluorescence of live cells and thepercentage of the PE positive cells were measured in conjunction withthe geometric mean of the positive population.

As shown in FIG. 13, the CAR9 and CAR6 CARs show a 10-fold highergeometric mean (21K for CAR9, 27K for CAR6) for EGFRvIII binding thanthe CAR10 (only 2K) when saturating amounts of EGFRvIII protein are usedfor detection, even though all constructs transduce equivalently (˜50%transduction efficiency for all). Similarly, the specificity for EGFR wtprotein is about 10-fold lower, as depicted by the log shift downwardsfor the staining with EGFR wt protein. This provides additional supportto the findings in the Jurkat cells above that indicate CAR9 and CAR6have a stronger affinity for EGFRvIII protein compared to CAR10 whenexpressed in primary T cells and suggest they will be more efficaciousin the clinic.

Functional analysis of the panel of humanized CAR constructs wasconducted as described in Example 8.

Example 8 Analysis of Humanized EGFRvIII-Specific CAR Constructs in TCells

To evaluate the feasibility of targeting EGFRvIII via a CAR technology,the humanized EGFRvIII scFv fragments were cloned into a lentiviral CARexpression vector with the CD3zeta chain and the 4-1BB costimulatorymolecule in two different configurations. The optimal construct isselected based on the quantity and quality of the effector T cellresponse of EGFRvIII CAR transduced T cells in response to EGFRvIII+ andEGFR wt targets. Effector T cell responses include, but are not limitedto, cellular expansion, proliferation, doubling, cytokine production andtarget cell killing or cytolytic activity (degranulation).

Materials and Methods

Generation of Jurkat Reporter Cell Line for Initial Characterization ofCAR Function

As an alternative to primary T cell transduction and activation, aJurkat-NFAT reporter cell line can be used to evaluate the functionalactivity of CAR constructs. The Jurkat T cell line (E6-1) wastransfected with a NFAT-luciferase reporter construct and a stable,clonal cell line (JNL) was selected for further characterization basedon strong induction of the NFAT reporter following PMA and ionomycinstimulation. The JNL cells are transduced with lentiviral vectors at aMOI of 5:1 and then expanded for 5-7 days. Before using in an assay, thepercentage of cells transduced (expressing the EGFRvIII CAR on the cellsurface) and their relative fluorescence intensity of that expressionare determined by flow cytometric analysis on an LSRII. From thehistogram plots, the relative expression levels of the CARs can beexamined by comparing percentage transduced with their relativefluorescent intensity.

Evaluating T Cell Activation of Humanized EGFRvIII-Specific CAR JNLCells

To evaluate T cell activation in the JNL reporter cell line, JNL orCAR-transduced JNL cells are plated at 50,000 cells per well in a 96well black plate with a clear bottom. Target cells (BHK parental cellsor BHK cells engineered to express either EGFRvIIII or EGFR wt) areadded to the wells to create effector to target (E:T) ratios of 1:2,1:1, 1:0.3, 1:0.1, 1:0.03, 1:01, and 1:0.003. PMA and ionomycin are usedas a positive control for activation. Cells are incubated at 37 C for16-24 hrs. At the end of the incubation, an equal volume of Bright-GloLuciferase assay reagent is added to each well. The plate is incubatedat room temperature for 10 minutes and then luminescence is measuredusing a luminometer.

Generation of Redirected Humanized EGFRvIII-Specific CAR T Cells

The humanized EGFRvIII-specific CAR lentiviral transfer vectors are usedto produce the genomic material packaged into the VSVg psuedotypedlentiviral particles. Lentiviral transfer vector DNA is mixed with thethree packaging components of VSVg, gag/pol and rev in combination withlipofectamine reagent to transfect them together in to 293T cells. After24 and 48 hr, the media is collected, filtered and concentrated byultracentrifugation or chromatography. The resulting viral preparationis stored at −80 C. The number of transducing units is determined bytitration on SupT1 cells.

Redirected EGFRvIII-specific CART cells are produced by activating freshT cells by engaging with CD3×28 beads for 24hrs and then adding theappropriate number of transducing units to obtain the desired percentageof transduced T cells. These modified T cells are allowed to expanduntil they become rested and come down in size (˜300 fl) at which pointthey are cryopreserved for later analysis. The cell numbers and sizesare measured using a Coulter multisizer III. Before cryopreserving,percentage of cells transduced (expressing the EGFRvIII-specific CAR onthe cell surface) and their relative fluorescence intensity of thatexpression are determined by flow cytometric analysis on an LSRII. Fromthe histogram plots, the relative expression levels of the CARs can beexamined by comparing percentage transduced with their relativefluorescent intensity.

Evaluating Cytolytic Activity, Proliferation Capabilities and CytokineSecretion of Humanized EGFRvIII Redirected CAR T Cells.

To evaluate the functional abilities of humanized EGFRvIII-specific CART cells to kill, proliferate and secrete cytokines, the cells are thawedand allowed to recover overnight. In addition to the humanizedconstructs, the murine CAR9 was used for comparative purposes whileSS1-BBz was used as non-targeting expressed CAR for background CAR/Tcell effect. For this flow based cytotoxicity assay, the target cellsare stained with CSFE to quantitate their presence. The target cellswere also stained for EGFRvIII expression to confirm similar targetantigens levels. The cytolytic activities of EGFRvIII CAR T cells aremeasured at a titration of effector:target cell ratios of 10:1, 3:1,1:1, 0.3:1 and 0:1 where effectors were defined as T cells expressingthe anti-EGFRvIII CAR. Assays were initiated by mixing an appropriatenumber of T cells with a constant number of targets cells. After 4 or16hrs, total volume of each mixture was removed and each well washed.The T cells were stained for CD3 and all cells stained with live/deadmarker 7AAD. After the final wash, the pelleted cells were re-suspendedin a specific volume with a predetermined number of counting beads. Cellstaining data was collected by LSRII flow cytometry and analyzed withFlowJo software using beads to quantitate results.

For measuring cell proliferation and cytokine production of humanizedCAR-EGFRvIII T cells, cells were thawed and allowed to recoverovernight. In addition to the humanized CAR-EGFRvIII, the murine CARSwas used for comparative purposes while SS1-BBz was used as anon-targeting expressed CAR for background CAR/T cell effect. The Tcells were directed towards U87, an astrocytoma-derived glioblastomacell line expressing or not expressing EGFRvIII. In addition, CD3×28beads were used to evaluate the potential of T cells to respond to thesecond round of endogenous immunological signals. To analyzeproliferation, T cells were stained with CSFE. The proliferation was thedilution of the CSFE stain reflecting the separation of the parentalmarkings now into two daughter cells. The assay tested only aneffector:target ratios of 1:1 and 1:0 where effectors were defined astotal T cells (CD4 and 8) normalized to express the anti-EGFRvIIIchimeric receptor at a common percentage. The assay was done induplicate and 24 hrs after mixing of the cells. Supernatant was removedfor cytokine production. After 5 days, T cells were stained forlive/dead with Live/Dead Violet (Invitrogen), then stained for CARexpression and phenotyped as either CD4 or CD8 cells. After the finalwash, the pelleted cells were re-suspended in a specific volume with apredetermined number of BD counting beads. Cell staining data wascollected by LSRII flow cytometry and analyzed with FlowJo softwareusing beads to quantitate results. Total cell counts were determined bynumber of cells counted relative to a specific number of beadsmultiplied by the fraction of beads yet to be counted.

Results

Jurkat Reporter Assay to Test the Ability of the Humanized CART-EGFRvIIICells to Recognize EGFRvIII Target Cells.

The ability of CART constructs to induce activation following targetengagement was measured with the JNL reporter cell line. The JNL cellline is engineered with a NFAT-Luciferase reporter construct which isinduced following target engagement of the CAR. JNL cells weretransduced with the various CAR-EGFRvIII constructs (CAR9, CAR3, CAR6,CAR8 and CAR10). Transduction efficiency was assessed by flow cytometryand was shown to be about 45-52% for all the constructs. TheJNL-CAR-EGFRvIII cells were then stimulated with seven different E:Tratios using three different target cells (BHK parental, BHK-EGFRvIII orBHK-EGFR wt). JNL parental cells and JNL cells expressing a control CARwere included as additional controls. The results in FIG. 14 showsignificant target induced activation can occur at ratios as low as1:0.01 for all constructs tested and CAR6 and CAR10 induce the mostactivation at the higher E:T ratios. No significant activation wasobserved with the EGFR wt expressing cells or by the control CARexpressing JNL cells. These data demonstrate specificity of the CARconstructs for EGFRvIII target and lack of cross-reactivity to EGFR wttarget.

Transduction and Expansion of Primary Human T Cells with the HumanizedEGFRvIII CAR Constructs

CD3+ T cells were obtained from apheresis products or whole blood fromhealthy donors. As described above, T cells were stimulated withCD3×CD28 beads for 24 hrs and then transduced with concentratedlentiviral supernatants at a MOI of 3. Cells were expanded in culturefor 8-10 days.

Cell surface expressions of humanized EGFRvIII CARs are comparable andtheir expression level very similar to murine CAR9. The overlay ofhistograms plotting the cell surface expression staining pattern of eachhumanized EGFRvIII-specific CAR transduced T cells and the meanfluorescent intensity (MFI) calculated from these profiles correlateswell with the percentage of cells transduced.

Proliferation Assay to Test the Ability of EGFRvIII Target Cells toStimulate Humanized EGFRvIII CART Cells

The ability of EGFRvIII specific CAR T cells to proliferate in responseto target engagement was evaluated in a proliferation assay.Subpopulations were enumerated by flow cytometry. Donor T cells weretransduced with either humanized CARs, murine CAR9 or SS1 (mesothelintargeting). CARs were mixed 1:1 or 1:0 with target cells and coculturedfor 5 days. FIG. 15 shows the ability of ND407 EGFRvIII CAR T cells toproliferate in an antigen specific manner. The dash bar indicates thenumber of T cells seeded and comparatively, no increase in T cellnumbers were detected targeting U87 while engagement with U87-EGFRvIIIinduced proliferation which was specific to EGFRvIII CAR T cellpopulations. The relative response for ND407 indicated that CAR6 andCAR8 are more robust than CAR9 or CAR3. The result of CD3×28 beadsindicates their stimulation was not enough to drive proliferation on asecond round of activation, similar to no stimulation at all.

ND407 T cells were used to screen different huEGFRvIII CARs for theirability to preferentially expand CAR+ T cells. FIG. 16 shows CARS andCAR6 consistently having the strongest CAR+ expansion in each donor.CAR+ increase is a result of a successful engagement with target,proliferation and survival of activation induced cell death of antigenrecognition.

Killing Assay to Test the Ability of the Humanized EGFRvIII CART Cellsto Kill EGFRvIII Target Cells

The ability of EGFRvIII specific CAR T cells to kill targets was testedin a Chromium release assay. The human glioblastoma cell line, U-87MG,was engineered to express either EGFR wild type receptor or the EGFRvIIImutant. These engineered cell lines served as the targets for thekilling assay. Three effector CAR T cells were used to determine thespecificity of killing target cells; 1) human T cells transduced toexpress murine 3C10 (CAR9), 2) human T cells transduced to express ahumanized version of the murine 3C10, referred to as CAR6 and 3) human Tcells transduced with a CAR specific for mesothelin, SS1. All effectorcells were normalized to express 30% CAR+ transduction. Target cellswere labeled with chromium-51 and washed immediately prior to coculture.The effectors and targets were mixed together at the indicated ratios(E:T) and allowed to incubate for 4 hours.

The results in FIG. 17 (A) shows that CAR T cells mixed with U-87 cellsexpressing the wild type EGFR receptor showed no cell killing abovebackground up to E:T of 50:1. However, the results in FIG. 17 (B) showthat in contrast, EGFRvIII specific CAR T cells, CAR9 or CAR6, mixedwith U-87 cells expressing EGFRvIII showed specific killing at E:Tratios of 6.25:1 up to 50:1. No significant killing was observed whenthe mesothelin specific CAR T cells were used as effectors. These datademonstrate the specific on target killing of target cells expressingEGFRvIII by CAR9 and CAR6 T cells, but no killing of cells expressingwild type EGFR or by a non-specific CAR T cell, SS1.

Cytokine Assay to Test the Ability of the Humanized EGFRvIII CART Cellsto Promote an Anti-Tumor Response and Demonstrate Specificity

The ability of EGFRvIII specific CAR T cells to induce cytokine inresponse to target engagement was evaluated in a co-culture assay. CAR Tcells were co-cultured with target expressing cells for 18-24 hrs atvarious target:effector ratios (0.3:1, 1:1, 3:1 and 10:1). Target cellsincluded U87 cells expressing the EGFR wt endogenous protein (U87 wt),U87 cells overexpressing EGFRvIII (U87-vIII), BHK (baby hamster kidneycells) parental cells, BHK cells overexpressing human EGFR wt protein(BHK wt), or BHK cells overexpressing human EGFRvIII protein (BHK-vIII).After 18-24 hrs, supernatants were removed from the cultures andcytokines analyzed using a Cytometric Bead Assay (CBA). The resultsclearly demonstrated that 1) CAR6 and CAR9 T cells induced similarlevels of IFNg in response to EGFRvIII-expressing cells and 2) thatneither CAR T cell population induced IFNg in response to EGFR wtexpressing cells. Importantly, these data together with the killing andproliferation data indicate CAR6 and CAR9 show functional specificityfor EGFRvIII and have the capability to promote an anti-tumor immuneresponse.

Example 9 Humanized Anti-EGFRvIII CART Cells Reduce Tumor Burden in Mice

Humanized anti-EGFRvIII CAR T cells were shown to reduce tumor burden invivo in mice. For example, #2173 (CAR6) humanized anti-EGFRvIII chimericantigen receptor (CAR) lentivirally-transduced human T lymphocytes weredelivered intravenously in xenogeneic immune-compromisedNOD/SCID/common-gamma chain−/− mice treated established U87vIII gliomatumors in vivo. Control mice with 5 day established U87vIII subcutaneousflank tumors receiving donor-matched non-CAR transduced T cells' tumorsgrew rapidly, both by direct subcutaneous tumor measurement usingcalipers (max length×max width), and by photon emission measured bySpectrum in vivo imaging system (IVIS). In treated mice receiving evenlow numbers (0.5-1×10⁶) of CAR6 transduced cells, tumor growth wasmarkedly reduced in mice in a dose-dependent manner.

In this example, 1×10⁶ U87vIII human gliomas expressing EGFRvIII,GFP+Luc+ were washed and injected subcutaneously in 100 μL saline in theflanks of 30 NSG immune-compromised mice (N=10/group). Human T cellswere stimulated with anti-CD3/28 coated beads and lentivirallytransduced with humanized EGFRvIII CAR scFv #2173 (CAR6). Followingtransduction, ex vivo expansion and bead removal, CAR transduced T cells(˜50% CAR+ by flow cytometry) were washed and injected in 100 μL salinevia tail vein 5 days after tumor implantation. Tumor growth wasevaluated by caliper measurement (upper left), and luciferin-inducedphoton emission (upper right). Measurements were started 7 days after Tcell transfer and 12 days after tumor injection. SEM is shown in FIG. 18(N=10 mice/group). Survival of each group is plotted by Kaplan Meiercurves in FIG. 18 (lower). All mice receiving control T cells died byday 26, with group receiving 0.5×10⁶ and 1.0×10⁶ CAR6 T-cells at 30% and90% survival, respectively as of experimental day 30.

Equivalents

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

What is claimed is:
 1. An isolated nucleic acid molecule encoding achimeric antigen receptor (CAR), wherein said CAR comprises ananti-EGFRvIII binding domain, a transmembrane domain, and anintracellular signaling domain comprising a primary signaling domain, acostimulatory domain, or both a primary signaling domain and acostimulatory domain, wherein the encoded anti-EGFRvIII binding domaincomprises an amino acid sequence with greater than 99% sequence identityto SEQ ID NO:68 comprising: (a) a heavy chain immunoglobulin variableregion comprising: (i) a CDR1 comprising the sequence DYYIH (SEQ ID NO:22); (ii) a CDR2 comprising the sequence RIDPENDETKYGPIFQG (SEQ ID NO:23); and (iii) a CDR3 comprising the sequence RGGVY (SEQ ID NO: 24); and(b) a light chain immunoglobulin variable region comprising: (i) a CDR1comprising the sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 26); (ii) a CDR2comprising the sequence LVSKLDS (SEQ ID NO: 27); and (iii) a CDR3comprising the sequence WQGTHFPGT (SEQ ID NO: 28).
 2. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid sequenceencoding the anti-EGFRvIII binding domain comprises a nucleotidesequence with greater than 99% sequence identity to SEQ ID NO:
 69. 3.The isolated nucleic acid molecule of claim 1, wherein the encoded CARcomprises a transmembrane domain that comprises a transmembrane domainof a protein selected from the group consisting of the alpha, beta orzeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 andCD154.
 4. The isolated nucleic acid molecule of claim 1, wherein theencoded transmembrane domain comprises the sequence of SEQ ID NO: 15; anamino acid sequence comprises at least one modification but not morethan 20 modifications of the amino acid sequence of SEQ ID NO:15; or asequence with greater than 95% identity to the amino acid sequence ofSEQ ID NO:15.
 5. The isolated nucleic acid molecule of claim 1, whereinthe nucleic acid sequence encoding the transmembrane domain comprisesthe sequence of SEQ ID NO:8, or a sequence with greater than 95%identity thereof.
 6. The isolated nucleic acid molecule of claim 1,wherein the encoded anti-EGFRvIII binding domain is connected to thetransmembrane domain by a hinge region.
 7. The isolated nucleic acidmolecule of claim 6, wherein the encoded hinge region comprises SEQ IDNO:14, or a sequence with greater than 95% identity thereof.
 8. Theisolated nucleic acid molecule of claim 6, wherein the nucleic acidsequence encoding the hinge region comprises the sequence of SEQ IDNO:7, or a sequence with greater than 95% identity thereof.
 9. Theisolated nucleic acid molecule of claim 1, wherein the encodedintracellular signaling domain comprises a costimulatory domain and aprimary signaling domain.
 10. The isolated nucleic acid molecule ofclaim 9, wherein the encoded costimulatory domain comprises a functionalsignaling domain of a protein selected from the group consisting ofOX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and4-1BB (CD137).
 11. The isolated nucleic acid molecule of claim 9,wherein the encoded costimulatory domain comprises the sequence of SEQID NO:16; an amino acid sequence which comprises at least onemodification but not more than 20 modifications of the amino acidsequence of SEQ ID NO:16; or a sequence with greater than 95% identityto the amino acid sequence of SEQ ID NO:16.
 12. The isolated nucleicacid molecule of claim 9, wherein the nucleic acid sequence encoding thecostimulatory domain comprises the sequence of SEQ ID NO:9, or asequence with greater than 95% identity thereof.
 13. The isolatednucleic acid molecule of claim 1, wherein the encoded intracellularsignaling domain comprises a functional signaling domain of 4-1BB and afunctional signaling domain of CD3 zeta.
 14. The isolated nucleic acidmolecule of claim 1, wherein: the encoded costimulatory domain comprisesthe sequence of SEQ ID NO: 16; an amino acid sequence having at leastone modification but not more than 20 modifications of the amino acidsequence of SEQ ID NO: 16; or a sequence with greater than 95% identityto the amino acid sequence of SEQ ID NO: 16, and the encoded primarysignaling domain comprises the sequence of SEQ ID NO: 17 or SEQ IDNO:99; an amino acid sequence having at least one modification but notmore than 20 modifications of the amino acid sequence of SEQ ID NO:17 orSEQ ID NO:99 or a sequence with greater than 95% identity to the aminoacid sequence of SEQ ID NO:17 or SEQ ID NO:99.
 15. The isolated nucleicacid molecule of claim 1, wherein the encoded intracellular signalingdomain comprises the sequence of SEQ ID NO: 16 and the sequence of SEQID NO: 17 or SEQ ID NO:99, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.
 16. The isolated nucleic acid molecule ofclaim 1, wherein the nucleic acid sequence encoding the intracellularsignaling domain comprises the sequence of SEQ ID NO:9, or a sequencewith greater than 95% identify thereof, and/or the sequence of SEQ IDNO:10 or SEQ ID NO:100, or a sequence with greater than 95% identitythereof.
 17. The isolated nucleic acid molecule of claim 1, furthercomprising a leader sequence.
 18. The isolated nucleic acid molecule ofclaim 17, wherein the leader sequence comprises SEQ ID NO:
 13. 19. Avector comprising a nucleic acid molecule of claim
 1. 20. The vector ofclaim 19, wherein the vector is selected from the group consisting of aDNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, and aretrovirus vector.
 21. The vector of claim 19, further comprising apromoter.
 22. The vector of claim 21, wherein the promoter is an EF-1promoter.
 23. The vector of claim 22, wherein the EF-1 promotercomprises the sequence of SEQ ID NO:
 97. 24. The vector of claim 19,wherein the vector is an in vitro transcribed vector.
 25. The vector ofclaim 19, wherein the nucleic acid sequence in the vector furthercomprises a poly(A) tail.
 26. The vector of claim 19, wherein thenucleic acid sequence in the vector further comprises a 3'UTR.
 27. Anisolated cell comprising the vector of claim
 19. 28. The cell of claim27, wherein the cell is a T cell.
 29. The cell of claim 28, wherein theT cell is a CD8+T cell.
 30. The cell of claim 27, wherein the cell is ahuman cell.
 31. A method of making a cell comprising transducing a Tcell with a vector of claim
 19. 32. A method of generating a populationof RNA-engineered cells comprising introducing an in vitro transcribedRNA or synthetic RNA into a cell, where the RNA comprises the nucleicacid molecule of claim
 1. 33. The isolated nucleic acid molecule ofclaim 1, wherein the encoded intracellular signaling domain comprises aprimary signaling domain.
 34. The isolated nucleic acid molecule ofclaim 1, wherein the encoded intracellular signaling domain comprises acostimulatory domain.
 35. The isolated nucleic acid molecule of claim 1,wherein the nucleic acid sequence encoding the CAR comprises thenucleotide sequence of SEQ ID NO:72, or a nucleic acid sequence havinggreater than 99% identity thereof.
 36. The isolated nucleic acidmolecule of claim 1, wherein the encoded CAR comprises the amino acidsequence of SEQ ID NO:73, or an amino acid sequence having greater than99% identity thereof.
 37. The isolated nucleic acid molecule of claim 1,wherein the encoded anti-EGFRvIII binding domain comprises the sequenceof SEQ ID NO:68.
 38. The isolated nucleic acid molecule of claim 2,wherein the nucleic acid sequence encoding the anti-EGFRvIII bindingdomain comprises the nucleotide sequence of SEQ ID NO:69.